HU228873B1 - Hcv antigen/antibody combination assay - Google Patents

Description

The present invention relates generally to viral diagnostics. More specifically, the present invention relates to an antigen / antibody combination assay for the accurate diagnosis of hepatitis C virus infection.

Hepatitis C virus (HCV) is the main cause of parenteral non-A, non-B hepatitis (NANBH), which is most commonly spread through blood transfusions and sexual contact. About 0.42.0% of blood donors are infected with the virus. Chronic hepatitis develops in about 50% of infections and liver cirrhosis develops in about 20% of infected individuals, sometimes leading to hepatocellular carcinoma. Accordingly, studying and curing the disease is of medical importance.

HCV was first identified and characterized by Houghton et al. As a pathogen of NA.N.8H. The genomic sequence of HCV is known, as are methods for its preparation [WO 89/04668; WO 90/11089 and WO 90/14463). HCV has a 9.5 kilobase, positive-sense single-stranded RNA genome, and is a member of the Flaviviridae virus family. Based on their phylogenetic properties, at least six but related genotypes of HCV have been identified (Simmonds et al., J. General Virology 74, 2391-2399 (1993)). The virus encodes a single polyprotein with more than 3,000 amino acid residues [Choo et al. al; Science 244: 359-362 (1989); Choo et al. (1991) Proceedings of the National Academy of Sciences USA 88: 245-1-2455; Han ·>

«* And others; Proceedings of the National Academy of Sciences, USA 33, 1711-1715 (1991)). Polyprotein is processed both during and after translation into both structural and non-structural (NSj proteins).

More specifically, as shown in Figure 1, the HCV genome encodes a number of proteins. The cleavage products and the nomenclature of the HCV polyprotein cleavage products are as follows; NHs-Bl-B2-C ~ p ~ ~ NS2NS3 NS4a-NS4b-NS5a-NS5b-C00H. The polyprotein initial cleavage is catalyzed by host cell proteases, which have three structural proteins, namely the N-terminal nucleocapsid protein (called "core") and two loop glycoproteins, also known as "E1" (also known as E), and release "E2M (also known as E2 / .NS1) and non-structural (NS) proteins containing viral enzymes. NS regions are designated NS2, NS3, NS4 and NS5. NS2 is a membrane protein of an integrase with proteotypic activity. NS2, either alone or in combination with NSS, drives the binding between NS2-NS3, which in turn generates the N-terminus of N33 and releases a large pobprotein that has both serine protease and RNA helperase activity. The NS3 protease serves to process the remaining polyprotein. Upon completion of the maturation of the polyprotein, autocatalytic cleavage of the NS3-NS4a bond is initiated by NS3 aaerin. catalyzed by protease. Subsequent cleavage of the HCV polyprotein NS3-a.s appears to involve recognition of the polyprotein cleavage sites by the NS3 molecule of another polypeptide. In these. In reactions, NS3 liberates an NS3 cofactor (NS4a), two proteins (NS4b and NSSa), and an RNA-dependent RNA polymerase (NB3b).

A number of general and specific pokpeptides derived from HCV polyproteins have been described as useful as HCV temunology and diagnostic reagents (Heughton et al., European Patent Nos. 313,216 and 383,232; Choo et al., Science 244: 359-392 (1939); Kuo et al. et al., Science 244: 362-364 (1989); Houghton et al. (1991) Hepatology 14: 331-388 (1991); Chien et al. (1992) Proceedings of the National Academy of Sciences USA 39: 100-110615; Chien et al. et al., 3. Gastroent, Hepatot 8, 333-39 (1993); Chien et al., International Patent Application 94/01773. These publications provide ample background for HCV in general and for the production and use of HCV polypeptide temxmologte reagents.

Sensitive, specific methods for screening and identifying HCV carriers and HCV-infected blood or blood products would represent important advances in medicine. Post-transfusion hepatitis (FTH) occurs in approximately 10% of transfused patients and in approximately 90% of these cases. The need for reliable diagnoses & prognostic tools for HCV, Patient care, and prevention of HCV transmission by blood or blood products, or close human contact Needs A number of essays have therefore been developed to serodiagnose HCV infection (Choo et al., Science 244, 359-392 (1989); Kuo et al., Science 244: 362-364 (1939); Choo et al. (1990) Br. Med. Bull. 46: 423-441 (1990); Ebeling et al., Chain 635: 932-983. Van Der Foel et al., (1991) 337: 317-319; Chien, DY: WO 94/01773; Valenzuela (1990); s' et al, International Patent No. WO 97/44469;

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Kawasakima et al .; 5,871,904. United States Patents].

In some serum-based assays, there is a significant problem that there is a significant gap between infection and detection of the virus, often exceeding 80 days. Bz examination gap may pose a high risk to blood transfusion recipients. To overcome this problem, nucleic acid-based assays (NAT | which directly detect viral RNA and HCV core antigen assays that directly test for vital antigen instead of antibody response have been developed [Kashiwakuma et al., U.S. Patent No. 5,871,904). Beid et al .; Transhxsicn 40, 575-579 (20000,

However, there remains a need for a sensitive, accurate diagnostic and prognostic tool to ensure appropriate treatment of patients and to prevent transmission of HCV to blood and blood products and close human contact.

The present invention is based in part on the discovery that HCV seroconversion antibodies are typically anti-nuclear and anti-NS3 (helicase) antibodies. Accordingly, the present invention provides a combination assay for HCV core antigen and NS3 antibody that can detect in a sample HCV antigens and HCV antibodies on a single solid matrix.

Thus, in one embodiment, the present invention provides an immunostaining solid support comprising at least one HCV anti-core antibody and at least one isolated HCV NS3 / 4a epitope bound thereto. The antibody and NS3 / 4a epitope can be any of the molecules described herein. In addition, the solid support * * φ * φ φ φ »

Φ «Ψ X {ί

X φ φ φ φ X «Φ Φ φ φ φ φ φ φ

Φ Φ Φ X Φ Φ φ φ φ φ φ *. * Φ * · ¹ φ φ may contain any of the epitope fusion antigens disclosed herein, such as the multiple epitope fusion antigen which represents the amino acid sequence shown in Figures 7A-7F. included.

In some embodiments, the solid support contains at least two HCV antigen core antibodies, bound before the anti.m.ag antibody may be a monoclonal efienak, the NS3 / 4a epitope may be a conformational epitope, e.g., the conformational NS3 / 4a epitope, which contains the amino acid sequence shown in Figures 4Ά to 4D.

In another embodiment, the present invention is directed to an immunomodulatory solid support comprising at least two HCV anti-core monoclonal antibodies and at least one HCV NS3 / 4 ' formitic epitope comprising the 4A-4D. The amino acid sequences shown in FIGS

In a further embodiment, the present invention relates to a method for detecting HCV infection in a biological sample. The method comprises the steps of providing a solid support for an immunoassay as described above; bj combining a biological sample with the solid support under conditions that allow HCV antigens and antibodies to be present. present in the biological sample, they bind to at least one anti-core antibody or to the HS3 / 4a epitope; ej complexing conditions, adding a first detectably labeled antibody to the solid support from step bj, wherein the first labeled anti-core antibody is directed against another HCV core epitope than at least one of the anti-core antibody bound to the solid support; iij an antigen that reacts with a KS3 / 4a epitope from a biological sample * X Φ ·> · * * *> X

Φ * X * * * * «>> 'X *« X

X «·» * »> · * w from HCV antibody; and H1) a second detectably labeled antibody, wherein the second detectably labeled antibody is responsive to the antigen of (ii); and d) detecting complexes between antibodies and antigens, if. there are some that indicate that biological tinintaphean is infected with HCV. HS3 / 4a. an epitope may be a conformational epitope, such as a conformational epitope shown in Figures 4A-4D. FIGS.

In a further embodiment, the present invention relates to a method for detecting HCV infection in a biological sample. The procedure consists of the following steps; a) providing an immunoassay solid support containing at least two HCV antibody antibodies bound as described above; b) combining a biological sample with the solid support under conditions that allow HCV antigens and antibodies, if present in the biological sample, to cure to at least one anti-core antibody or NS3 / 4a epitope; c) under tonplex imaging conditions, adding to the blunted carrier from step b) i) a first detectably labeled antibody, wherein the first labeled anti-antibody is directed against another HCV core epitope, not against the solid-carrier anti-core antibody; ii) an epitope derived from the c33c region of the HCV polyprotein fused to an hBOD amino acid sequence; and III) a second detectably labeled antibody, the second detectably labeled antibody reacting with the amino acid sequence hSÖD; and d) curing the complexes formed between the antibodies and the antigens, if any, which indicate that the biological sample is infected with HCV. Epitope H33 / 4a can be a conference:

ΧΦ Φ φ φ φ

Φ Φ ν Φ ¥ ΦΦ

Φ Φ Φ

Ep Φ Φ'Φ, such as the conformational epitope having the HS3 / 4a sequence shown in Figure 4A-41X.

In any of the foregoing embodiments, the anti-core antibody may be directed directly against an N-terminal region of the HCV core antigen, such as the HCV 10-S3 amino acids, numbered according to the HCV1 polyprotein sequence, and / or detectably. labeled HCV antb core antibody may be directed against a C-terminal region of the HCV core antigen, such as HCV 120-130 amino acids, numbered according to the HCV1 polyprotein sequence. In addition, the antigen that reacts with the HCV antibody in the biological sample may be derived from the NS3 region, e.g., an epitope of the c33c region of the HCV pcprotem and fused to a human smpewmd dismutase (hSÖD) sminic acid sequence. In this embodiment, the. a second, detectably labeled antibody reacts with the hSOD amino acid sequence

In another embodiment, the present invention provides a method for detecting HCV infection in a biological sample. The procedure consists of the following steps: providing a immunostaining solid support containing two HCV anti-core monoclonal antibodies and a conformational epitope containing the 4A-4D. FIG. head combined with a biological sample with a solid support, conditions that allow HCV antigens and antibodies, if present in the biological sample, to bind to at least two anti-core antibodies and the HS3 / 4a conformational epitope; under the head step sdlrd .hcrdoschcz adds comptent conditions * · *> ·> V>

* Μ * <X * «· * ♦ -W ΦΦ don (i) a detectably labeled antibody, the first being detectable. labeled antibody is a detectably labeled HCV antibody and the labeled antibody is directed directly to another HCV core epitope other than at least two antibody bound to the solid support, H> an epxtope from the o33c region of the HCV polyprotein, linked to a major amino acid sequence; and iii> a second detectably labeled antibody, the second detectably labeled antibody reacting with said hSÖD amino acid sequence; then detecting the complexes formed between antibodies and antigens (if any) "suggesting" that the biological sample is infected with HCV

In some embodiments, the. at least two antimicrobial antibodies are directed against an N-ternary region of the HCV core antigen, e.g., HCV amino acids 10-53, according to the numbering of the HCV polyprotein, and the detectably labeled HCV antigen antibody is a C- against the terminal region of the HCV, such as amino acids 120-130-aa of HCV, according to the numbering of the HCV polyprotein.

In another embodiment, the present invention provides a method for detecting HCV infection in a biological sample. The method comprises the steps of: a) providing a solid immunosuppressive carrier comprising multiple epitope fusion antigens;

b) combining a biological sample with the solid support under conditions that allow the HCV antigens and antibodies, if present in the sample, to bind to at least one anti-nuclear antibody »to the opitope HS3 / 4a and multiple an epitope for Wt antigen; o) adding the solid support obtained in step b) under complexing conditions. ii) a first detectably labeled antibody directed against another HCV core epitope other than at least one antigenic antibody bound to the solid support; ü) first and second antigens which react with the HCV antibody in the biological sample, which is capable of reacting with the NS3 / 4a epitope and the multiple fusion antigen; and providing a second detectably labeled antibody in which the second detectably labeled antibody responds to the health of step (i); (d) detecting antibodies, if any, between the antibodies and the antigens and indicating that the biological sample is infected with HCV.

The anh-core antibody may be directed against the N-terminal region of the HCV anthmag antigen, and said first detectably labeled antibody may be directed against a C-terminal region of the HCV core antigen, as described above. In addition, the first antigen, and reacts with the HCV antibody in the biological sample, may contain an antibody from the eS3e region of the HCV polyprotein, and may fuse with a bSÖD amino acid sequence. In this context, the second detectably labeled antibody reacts with the hSÖD andnoasv sequence. In addition, the. a second antigen that reacts with an HCV antibody in the biological sample may contain an epitope from the c22 region of the HCV polyprotein, such as the Lysw-Sem amino acid.it of the HCV polyprotein, Arg4? and substitution of heu for Trp at position 44, numbered based on the HCV polyprotein sequence. The epitope may be fused to an hSOD amino acid sequence. If so, the antibody that is labeled as khutatbase reacts with the amino acid sequence hSOD. The multiple-fusion antigen may contain 7A-7F. 3A to 4B.

* «Χ * X χ« χ · x- ♦ χ χ «« x x. «· ♦ · * *» * «« «X *** x *« «x«

In a further embodiment, the present invention relates to a method for detecting HCV infection in a biological sample comprising the steps of: a) providing a solid immunostaining medium containing two HCV anime monoclonal antibodies, a conformational epitope of HCV NS3 / 4a, 4A-4D, and bound to a multiple epitope fusion antigen having the sequence set forth in Figures 7A-7F; b) combining a biological sample with the solid support under conditions that allow the HCV antigens and antibodies, if present, to bind to at least two anti-core antibodies, the HS3 / 4a conformational epitope, and the multiple epitope fusion. antigen; ej adding to the solid support obtained in step b) under crosslinking conditions 1) a first detectably labeled antibody which is a detectably labeled HCV anti-core antibody and the labeled anti-core antibody is directed against another HCV core epitope, at least two anti-core antibodies bound to the solid support; ii) an epitope of the e33e region of the HCV polyprotein fused to an hSOD amino acid sequence and an epitope of the c22 region of the HCV polyprotein fused to an hSOD amino acid sequence; and iii) a second detectably labeled antibody, said second detectably labeled antibody reacting with said hSOD amino acid sequences; d) detecting the compote formed between the viruses and the antigens, if present, which indicates that the biological sample is infected with HCV.

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* ♦

In this embodiment, the at least two anti-core antibodies may be directed against an N-terminal region of the HCV core antigen, such as HCV amino acid residues 10-53, numbered according to the HCV1 polyprotein, and the detectably labeled HCV antibody core is directed against the C-terminal region of the HCV core antigen, such as HCV 12-13G, numbered according to the HCV1 polyprotein. In addition, the c22 region may contain the Lysio ~ Ser <? 9 amino acids of the HCV polyprotein, by excision of Arg47 and its replacement with Len Trp at position 44, numbered based on the HCV1 polyprotein sequence.

In other embodiments, the present invention provides kits for immuno-diagnostic assays comprising an immunoassay solid support as described above, and instructions for use of the immunoassay assay. In another embodiment, the present invention provides methods for preparing an immunoassay solid support that comprises: comprising the steps of: a) providing a solid support; and b) binding of at least one HCV anti-core antibody, i.e. one, two or more, and the NS3 / 4a epitope of at least one isolated HCV, and optionally a multiple epitope fusion antigen. Anti-core antibodies, NS3 / 4a epitopes, and multiple epitope fusion antigens have been described above.

In still other embodiments, the present invention provides a multiple epitope fusion antigen that is a 7A-7F. 1 to 4, or an amino acid sequence having at least 80% sequence identity, such as 90% or greater, that specifically responds to an anti-HCV antibody in a biological sample from an individual infected with HCV. HCV Antibodies In some embodiments, the multiple epitope fusion antigen is 5A-SF. is the amino acid sequence shown in FIGS.

In yet other embodiments, the present invention provides a potinuRieotide comprising the coding sequence for a multiple epitope fusion antigen as described above, recombinant vectors comprising the polymerMeotides, host cells transformed with recombinant vectors, and the like. methods for producing a recombinant multiple epitope fusion antigen, the method comprising the steps of: a) providing a population of cells as mentioned above; and

b) culturing a population of cells under conditions such that the multiple epitope fusion encoded by the coding sequence in the recombinant vector is overexpressed.

These and other aspects of the present invention will be apparent from the following detailed description and accompanying drawings.

The accompanying figures are briefly described below.

Figure L is a diagrammatic representation of the HCV genome depicting the various regions of the polyprotein from which the antigens (proteins and antibodies) of the assay of the present invention are derived.

Figure 2 is a schematic representation of a representative antibody / antigen combination assay of the present invention,

Figure 3x depicts a representative NS3 / 4 & conformation antigen amino acid used in the assays of the present invention.

ϊ acid sequence. The iron holder! alanine replaces it at position XS2 as normally found at that position.

A. 4A-4D. Figure 4A-4D depicts the antigenic DNA and corresponding amino acid sequence of a representative NS3 / 4a conformation used in the assays of the present invention. FIGS. The amino acids at positions 403 and 404 indicate that, relative to the natural amino acid sequence of HCV-1, Thr is Pro and Ser is He.

Figure 5 is a diagram of the preparation of pd, HCVla.nx3ns4aR. Figure 6 is a 12-diagram representation of MBFA.

7A-7.P. Figure 12 shows the DNA sequence of MBFA 12 and its corresponding amino acid sequence;

Figure 8 is a schematic diagram of a representative immunoassay of the present invention using »ΜΕΡΑ12

In the practice of the present invention, unless otherwise noted, the conventional methods of chemistry, &quot; biochemistry, &quot; recombinant DNA techniques and immunology, &quot; are known to one of ordinary skill in the art. These techniques are described in detail in Pundamental Virology, 2nd Edition, p. and II.

Fields and D.M. Krtipe; Handbook of Bzperimental Immunology, Volume I, Thousand

ML Weir and C.C. Blactovell »

Scientific Riblications; T. B. Creighten, Profems, Structures and Moleoular Properties (W.H. Freeman and Company, 19931; A.U. Lehninger, Hiochemistry (current publication by Wortb Fubiishers Inc.); Sambrook et al., Molecnlar Clomng;

U <O mm, ϊ na s av m

Φ «ς. χχ.

Cola Spring Harbor Press, Cold Spring Harbor, N.Y. (1989); Methods in Biology, edited by S. Colowick and TC Kaplan, Academic Press Inc.).

It should be noted that in this specification and the appended claims, the singular grammatical terms have the same meaning. book a. unless the context requires otherwise. Thus, for example, "a reference to an antigen includes a mixture of two or more antigens, and the like.

The following abbreviations are used throughout the text:

Alaním Alá (B) Asparagine: Aon (N | Cysteine: Cys (C) Clutamic Acid Gin (E) Hisatcline: His (H) Lenem; Len (L) Melionin: Mei (M) Prolin; Pro (P) Threonine; Thr. (T) Tiroaln; Tyr (¥)

Ax'ginin: Arg (R) Asparagic Acid: Asp (D) Glotamine: Gin (Q) Glidn: Gly (G) Flavor: He fi) lian; Lys (K) Penllalanine: Phe (P) Serine: Ser (S) Tryptophan: Trp (W) Valin: Val (V)

In the description of the present invention, the following terms are used and are intended to be defined as follows:

The terms "polypeptide" and "white" refer to a polymer of amine acids and are not corrected. for a .minimum length of the product. Thus, &quot; de &quot; refers to peptides, oligopeptides, dimers, multimers and the like.

The prior art includes modifications of the polypeptide after expression, such as ghcosylation, aesthetization, stretching and the like. In addition, the present invention includes a term such as "polypeptide *" natural sequence. modifications, such as deletions, additions and substitutions (generally conservative substitutions), as long as the protein retains the desired activity. These. modifications may be intentional, such as site-specific amphagenesis, or they may be random, such as mutations in the host protein producing proteins, or due to errors in the polymerase chain reaction. It may be derived from HCV but may be produced synthetically or by rocombinant methods. In addition, the polypeptide may be derived from any of the various strains of HCV, such as, for example, HCV Immune 2, 3 or 4. Among these strains, many conserved and variable ancestors are known, and generally the amino acid sequences of epitopes derived from these regions exhibit a high degree of azequoene homology, i.e., a homologue of more than 30% of the amino acid sequence, preferably more than 40% homologue. under each other. Thus, for example, the term _s MS3 / 4a * polypeptide refers to native HS3 / 4a from any of the various HCV strains, as well as to HS3 / 4a analogs, muteins and immunogenic fragments, as further defined below. These hands on strains are known to have more than one known genotype (B, 160.08?

U.S. Pat. GenBank AJ238800 and AJ'238799 Deposit Numbers).

The terms "analogue" and "artificial" refer to biologically active derivatives of the reference molecule, or fragments thereof, which are retained in the assays herein. desired activity, such as immune reactivity. The term "analogue" generally refers to a compound having the natural polypeptide sequence and structure, addition, substitution (usually conservative substitution), and / or deletions of one or more amino acids relative to the natural molecule, as long as the modifications do not impair the immunogen. The term "rnutein" refers to peptides having one or more peptidomimetics ("peptidoids"), such as those disclosed in WO 91/04282. Preferably, the analogue or its lysine has the same immune activity as the natural molecule. Methods for making polypeptide analogs and muteins are known in the art and are described below.

Particularly preferred analogs include substitutions that are conservative in nature, i.e., substitutions that are anti-side chain based within amino acid families. More importantly, amino acids are generally classified into four families: 1) acidic - aspartate and glutamate; 2) basic lian, arginine, histidm; 3) apolar - apple, valine, linseed, .isoleudn, proline, phenylalanine, methanamine, tryptophan, and 4) unpolarized polar - glycine, asparagine, glutamine, distem, serine, threonine, and tiroaln. Fendalanine, tryptophan and tyrosine are classified as slightly aromatic amines. For example, it's reasonably boiling; "

ΦΦ ί

it is believed that the substitution of leucine with feo.lend.nnol or vair, an aspartate gututamate, a threonine azerine, or a similar conservative substitution of an amino acid for a structurally related amino acid will have little effect on biological activity. For example, the Pohpeptide of interest may contain 5-10 conservative or non-conservative amino acid substitutions, or even 15-25 conservative or non-conservative amino acid substitutions, or 5 to 25 · any number of substitutions as long as the desired function of the molecule remains intact . One of ordinary skill in the art can easily determine the ancestors of a molecule of interest that are able to tolerate change, using well-known Hopp / Woods and Kyte-Doolittle representations in the art.

food, which contains the intact, full length, p and so portion

the. a C-terminal grant and / or N-terminal deletion of the natural polypeptide. The 4mmunogenic fragment of a particular HCV protein will generally contain at least about 5 to 10 contiguous amino acids from the full length molecule, preferably at least about 15 to 25 contiguous amino acids from the full length molecule, and most preferably at least about 1 or amino acids from the full length molecule. epitope, or an integer number of amino acids between 5 and the number of amino acids in the full-length sequence, provided that said fragment retains its immunoreactivity in the assays described herein. For example, the preferred immunogenic fingη φ * is also considered to be mens, without being limited to gunfcat today, a. The HCV core has more common amino acid residues 10-45, 10 ~ 53, 7 ~ 88 ~ aa and X2 ~ X3O ~ of the polyprotein, the 5-1-1 epitope (in the HS3 region of the viral genome). ) as well as the HCV protein. Defined epitopes from the E1, E2, c33c (N33), ct06 (NS4), HS3 / 4a and NS5 regions, and any of the various epitopes identified in the HCV polyprotein (Chien et al; Proeeedings of the National Academy of Sciences, VSA 89, 10011). (1992) 10015; Chien et al., D, Gastroent Hepatol 8, 333-39 (1993); Chien et al., WO 93/00365 to Hamaetkozi; Chien, DX: WO 94/01773; U.S. Patent Nos. 6,150,037 and 6,421,020 to Ahaxnok).

As used herein, the term "epitope *" refers to a sequence comprising at least 3-5, preferably about 5 to 10 or 15, and about 1000 (or their whole salts thereof) amtnesic acid, which defines a sequence which in itself, or as part of a naob obh sequence, it binds to an antibody generated against this sequence. There is no critical upper length of the fragment, which may contain nearly full-length lobe sequences, or it may be a fusion protein that has two or more epitopes from the HCV polyprotein An epitope useful in the present invention is not restricted to a polypeptide having exactly the same sequence as a sequence fragment of the parent protein from which it is derived. In fact, viral genomes are constantly changing and contain a number of variable domains that show a relatively high degree of variability between isolates. Thus, the term "epxtop *" refers to sequences which are identical to the natural sequence and also to modifications of the natural sequence, e.g. and additions (generally of a conservative nature).

Regions of a particular polypeptide containing an epitope can be identified by any of several epltop mapping techniques well known in the art (Glott E. _t Morris, Humana Press, Totowa, New Jersey, Ed. 66). For example, linear epitopes can be determined by simultaneously synthesizing a large number of peptides on solid supports that correspond to parts of the white molecule, and then reacting the peptides with antibodies while the peptides are still bound to the carrier. techniques are well known in the art (U.S. Patent No. 4,708,871 to Geysen et al., 1984, Froceedings of the National Academy of Sciences, USA 61, 3998-4002; Geysen et al., Froceedings of the National Academy of Sciences, 1984). USA 82: 178-182 (1985); Geysen et al. (1986) Molec. Immunot. 28: 709-715]. several epitopes of HCV have been identified (see, e.g., Chien et al., 1994, Yiral Hepatitis and Liver Disease 320-324; Similarly, conformational epitopes can be readily identified by the spatial conformation of amino acids, such as RNntgen crystallography and two-dimensional nuclear magnetic resonance (Bpltpp Mapping-Protocols, Methods in Molecular Bielogy, Ed. Press, Totowa, New Jersey (1996)). A φ φ

Φ Φ X ς

Φ * Φ φ φ>

** Φ ΦΧ · antigenic proteins of proteins can also be identified using standard annealing and hydropathic curves, such as those calculated using the Omiga LO program available from Oxford Molecular Group This computer program uses the Hopp / Woods method to determine the antigenicity profile (Hopp et al. (1981), Proceedings of the National Academy of Sciences, USA 78, 3824-3328), and use the Kyte-Doolittie technique to determine the hydropathic curves (Kyte et al., 1932, Journal of Molecular Biology 157, 103-132). ).

Hereafter referred to as a portion of the full-length term of the Jconformadic epitope *, or an analogue or synthetic thereof having the same native structural properties as the amino acid sequence encoding the epitope in the full-length natural protein. to limit ourselves to these, a. glycogenicity and three-dimensional structure. The length of the epitope-defining sequence may vary within sharp limits, since these epitopes are thought to be generated by the three-dimensional shape of the antigen (i.e., blackening). Thus, the number of amino acids defining the epitope may be relatively low but spread over the length of the molecule. on different molecules), and these parts are brought to the correct epitope conformation by blackening. For example, the deletion or substitution of these interrupting sequences does not necessarily affect the conformation of the epitope, as long as the conformation of the epitope is not critical (

XX * (for example, dysteines that play a role in disulfide bridges, ghcosylation sites, etc.).

The conformational epitopes in the N83 / 4a region can be readily identified by the methods described above. In addition, the presence or absence of a conformational epitope on a particular polypeptide protein can be readily determined by comparing the antigen of interest with an antibody (polyclonal serum or monoclonal antibody specific for the conformation epitope) and reactivity. reactivity of the denatured version of the antigen, which retains only linear epitopes (if any). In such an azeolysis using polyclonal antibodies, it may be advantageous to pre-absorb the polydonal serum with the denatured antigen to see if it retains antibodies to the carbon atom antigen. In addition, in the case of N83 / 4a, a molecule that retains its natural conformation may additionally have protease and optionally heucase endmactivity. Such activities may be determined by enzymatic assays as described below.

Preferably, a conformational epitope can be produced by recombinant methods and expressed in a cell, extractable from ara, under conditions that retain the desired structural properties, such as without denaturation of the epitope Such cells include bacteria, yeasts, insects and mammals. cells. The expression and isolation of recombinant HCV polyprotein recombinant epitopes have been reported in the literature (see, for example, WO 96/04301, WO 94/01773, WO 95/3303, WO 92/08734). In another version, the antigens are · * '>

Φ ΧΛΦ

Φ * «

* Φ Φ X * · Φ Φ .Φφ ΦΦ can be spliced and then, after recovery, the protein can be renaturated. It is also clear that chemical synthesis can produce conformational antigen mimotopes that cross-react with the conformational epitope of the "natural * antigen".

The term "multiple epitope fusion antigen *" or "MEFA *" hereinafter refers to a polypeptide in which multiple HCV antigens are part of a single continuous amino acid chain that does not occur in nature. HCV antigens may either be directly linked by peptide bonds or may be separated by stone-fusing amino acid sequences. Fusion antigens may also contain exogenous sequences relative to the HCV polyprotein. In addition, the HCV sequences present may be derived from multiple HCV genomes and / or descendants. The use of each MEFA in the thousandth immunoassay of the present invention is described in the literature (see, for example, WO 97/44469) and will be further described.

The term "antibody" refers to a molecule that binds chemically or physically to a polypeptide of interest, such as an HCV core antibody is a molecule that specifically binds to the HCV nuclear fetus. The term "antibody", as used herein, refers to an antibody that can be prepared from either a pclonal or a monoclonal preparation, and from the following: hybrid (chimeric antibody molecules (see, e.g., Winter et al; Naturre 349: 293-299 (1991b), S16, S6). F {ab> and F (ab> ptagments; W molecules are non-covalent hetem dimers; see, e.g., Inbar et al., U.S. Pat.

·>

Μ.

ϊ

$ XX ·

Froceedings of the National Academy of Sciences, VSA 69: 26592662 (1972); Rbrüch et al; Biochemistry 19: 4091-4096 (1980}}; single chain Fv molecules (sFv) (see, e.g., Muston et al., Boceedings of the National Acadcmy of Sciences, USA 85: 5879-5883 (1988}}; dhner and trimeric antibody fragment constructs »mini antibodies (see, for example, Fack et al., 1992, Biochemistry 31, 1.579-1584; Oumber et al., J. of Immunol. 140). 120-126 (1992); humanized antibody molecules (see, e.g., Riechmann et al., 1988, Nature 383, 323-327; Verhoyen et al., Science 939, 15344536 (1988); GB 2,276,169, published 1994). and on any of the fusion-derived fragments derived from these molecules, which retains the immunological binding capacity of the parent antibody molecule.

Hereafter, the term "monoclonal antibody" refers to an antibody composition that is a homologous antibody population. The term is not limited to the species or source of the antibody nor does it limit the method by which it was produced. Thus, the term includes antibodies derived from hybridomas as well as human monoclonal antibodies produced from human rather than rodent bifferidoma (see, e.g., Cote et al., Monodonal Antibodies and Cancer Therapy, p. 77).

The recombinant protein is a protein that retains its desired activity and has been produced, as described herein, by recombinant DNA technology. The gene of interest is usually timed and then expressed in the transformed organism as described below. The host organism expresses the foreign IUx gene, producing protein under enpressive conditions.

The term Penlalr, when it refers to a poHpeptide, means that the molecule in question is separated from the entire organism in which the molecule is naturally occurring or substantially free of other biological macromolecules of the same type. means that the nucleic acid molecule is free, partially or completely, of the sequences with which it occurs in nature; or a naturally occurring form of the sequence but linked to heterologous sequences; or a molecule that is disassembled from the chromosome.

The term &quot; equivalents of antigenic determinant * refers to an antigenic determinant derived from various strains or subspecies of HCV, such as HCV X, 2 or 3. More specifically, epitopes are known, such as 5-1-1, and such epitopes differ in strains 1, 2, and 3. Thus, 5-1-1 epitopes from three different strains are equivalent antigenic determinants and thus "replicas *, even if their sequence is not the same. In general, the amino acid sequences of the equivalent antigenic determinants have a high degree of sequence homology, i.e., greater than 30%, preferably greater than 40% amino acid sequence homology when the two sequences are aligned.

The term &quot; homnlngbü &quot; refers to percent homology between two polynucleotides or polypeptide units, seven DHS or two polypeptide sequences being substantially homologous to each other if the sequences are at least about 50 percent, preferably at least about 75%, more preferably at least . » * · ♦ <«· * <

Φ <<.. X X · * · * * * * «« «χ χ χ χ χ χ S S S S S S S S S S S S S S S S S S S. Subsequently, the term "substantially homologous" refers to sequences which show complete identity to the specified DNA or polypeptide sequence.

The term "identity" generally refers to the exact nucleotide or amino acid to amino acid correspondence between two polynucleotide or two polypeptide sequences. Percent identity can be determined by directly comparing the sequence information of the two molecules, by aligning the two sequences, counting the exact morph codes between the two aligned sequences, dividing it by the length of the shorter sequence and then multiplying by 100.

Easily accessible computer programs can be used to analyze similarity and identity, such as AL1GN fDayhöS, MO: ih: Atlax of Protein Sequenco and Structure, 5, Suppl 3, 353-358 xzsrkz MO, Dayhoff, National Biomedical Research Foundation, Washington DCj. which uses the algorithm of Snhth and Waterman's local homologue (Advances In Appt Mafla 2, 482-489 (1981)] for pepdd analysis. Programs for determining nucleotide sequence similarity and identity are provided in the Wisconsin Sequence Analysis Package ~ (8). v «w) are available (Genetic Computer Group, Madison, WI), such as BBSTFTT, FA8TA, and GAP, which are also based on the Smith and Waterman algadmus. parameters, which are described above, Wiseonsm Saganuáo Malyoh Pacfcage-fecn For example, a> * * «Ψ * x * *» IS í «S ** given n The percentage similarity of the ukleodd sequence to a reference sequence can be determined using the Smith and Waterman homology algorithm, with a base table of values, and a gap opening calculation for six nucleotide positions

Another method for determining the percent similarity of the present invention is to use the MPSRCH suite (copyright Iteivemfy of Edinburgh, developed by John F, Colima and Shane S. Turrock, distributed by IntelkGenetics, Inc. (Mountain View, CA}}). The SmithWaterman algorithm can be used, using the basic parameters for the value table (for example, a gap opening penalty of 12, a gap clearance penalty of one, and a gap effect of six.) From the data obtained, the fit * reflects "sequence similarity". suitable programs are also known for calculating percent identity or similarity of sequences, such as BLAST, using basic parameters, for example, BIÁSTH and BIASTF can be used using the following basic parameters: genetic code standard; number>both; cut value ^ night; v &rb-lo; Ma trix ^{: s!} BhÖSUM62; insert ^ SÖ sequence; classify HIGH VALUES by; Databases ^ non-redundant, Gene, Bank * EMBL * DDBd * FDB * Gen, Ba.nk CDS Translational * Swiss Protein * Spupdate * Fl, R. The application dimensions for these programs can be found at: bttpte / SlhFdfaMm / áBb

Alternatively, the homologue may be constructed based on the nucleotide sequence of the nucleotides under conditions that result in stable duplexes between ^the homologous regions, followed by single-stranded digestion with sped fluctuating enzymes, and determination of the size of the digested fragments, Substantially homologous DNA sequences can be determined in a Southern biot hybridization assay, for example under stringent conditions to be selected for the particular system. Selection of the appropriate hybridization conditions is within the skill of the art fSambrook et al. Molecular Cloning: A Laboratory Manual; 2nd edition, Cold Spring Harbor Press, Cold Spring Harfeor, NX (1989), DNA Cloning, Nucleic Add Hybrids. supra).

The Bgy coding sequence is ⁰ , or a sequence encoding a selected protein, a nucleic acid molecule that is transcribed (in the case of DNA) and translated (in the case of mRKS) into a polypeptide, m customs or edited when under the control of appropriate regulatory sequences. The coding sequence is bounded by a startodon at the 5 * (amino) end and a translational stop codon at the 3 * (carboxy) end. A trans-terminus-terminated aze sequence may be at the 3 'end of the coding sequence,

The term "x-linked *" refers to the arrangement of elements in which the components described herein are configured to perform their intended function. Thus, a given promoter is operably linked to a coding sequence if it performs transcription factors, sth, are present. The promoter should be eugenic with the fetal escape sequence as long as it functions to control its expression. , such as ϊ oc: »♦ *» »« j »« «* <χ» W »♦ *, and the promoter sequence can still be considered as" operably linked "to the coding sequence.

The Jcontroli element * term is one. The term may refer to promoters, transcriptional terminal sequences, upstream regulatory domains, polyadenylation signals including 5'-UTR-e.kef and 3 '~ UTBs, and ha. , then the leader sequences and enhancers, which together provide for the transcription and translation of a coding sequence in a host cell.

The term "promoter" is hereinafter referred to as a DNA regulatory region capable of binding a gaxdaseftheo to RNA pofimerase and initiating the transcription of a downsfmam (3-way) artificial coding sequence thereof. For purposes of the present invention, a premoter sequence is included. the minimum number of bases or elements required to lyse transcription of a gene of interest is within the promoter sequence of the transcript initiation site within the promoter sequence, as well as the protein binding domain (consensus sequence) that binds RNA. Eukaryotic pmmoters often, but not always, contain "TATA® fetchers and" OAT hoxo

A control sequence "directs" the transcription * of a coding sequence * in a cell when the RNA polymerase binds to the promoter sequence and transcribes the coding sequence into an mRNB and then translates it into a polypeptide encoded by the coding sequence.

«X» t Í4.

X << · * x ** * * * a * <ί · χ * * * ·>

"*". .j »· χχ

The term "expression cassette®" or "expression construct" refers to a composition capable of controlling the expression of sequences or genes of interest. The expression cassette contains control elements as described above, e.g., a promoter operably linked to the one of interest to us. chair gene (s), or gene (s) (to control their transcription), and often also contain polyadenylation sequences In some embodiments of the present invention, the expression cassette described herein may be in a plasmid construct, and in addition to the components of the expression cassette. one or more aze marker, i.e., a signal that allows the plasmid construct to exist in the form of single-stranded DNA (e.g., an M13 origin of replication) and contains at least one cloning site, and a "mammalian * replication origin (such as a SV4O or adenovirus replication origin),

"Transformation®", as used herein, refers to the introduction of an exogenous pobnucleotide into a host cell, "irrespective of the method of administration: for example, transformation by direct uptake, transfection, infection and the like. See below for details of the transfection methods. The exogenous polynucleotide may persist as a non-integrating vector, such as an episode, or alternatively may integrate into the host genome.

The term "cellular cell *" refers to a cell that is trmxformed or can be transformed with an exogenous DNA sequence.

The "common solid support" refers to a solid matrix to which .HCV polypeptides used in the immunoassay are covalently bound or non-covalently bound, such as by hydrophobic adsorption.

The term "immunologically reactive" means that the antigen in question specifically reacts with anti-HCV antibodies in a biological sample from an individual infected with HCV.

The term "immune complex" refers to the combination that results when an antibody binds to an epitope on an antigen.

Hereinafter, "biological sample *" shall mean a sample of tissue or fluid isolated from a subject, including but not limited to, blood, plasma, serum, stool, urine, bone marrow, bile, spinal cord. adipose, lymphatic fluid, skin samples, substances secreted by the skin, respiratory tract, digestive tract, and prostatic organs, tears, saliva, milk, blood cells, organs, a. biopsies and in vitro cell culture components including, but not limited to, conditioned media from cells and tissues, such as recombinant cells and cellular components, in culture media.

Hereinafter, the term * and «detectable label * shall mean a molecule that can be detected, including, but not limited to, radioactive isotopes, omissions, chemiluminescals, chromophores, entemefest, entem-substrate, entem-cotector, oz enrimite, chromofem-

ί »** ** · *« ί kai, dyes, metal ions »the fémkoUoidokot, ligands (e.g. feiotint the sztrepfavídint or haptens) and the like Λ ^ Onoreazkáló parent ^ * refers to a substance or refers to a portion »capable of Ouorescence in the detectable range. Examples of labels that can be used in the present invention include, but are not limited to, horseradish peroxidase (HRF), "Ouorescein", "fluorescein, rhodamtn, dansyl", "umbelliferone", "dimethyl acridinium ester (DMAB)," Texas red ", .Images »NADFH and α-β-

Before describing the present invention in detail, it should be noted that the present invention is not limited to certain formulations, or "parameters," as these may, of course, vary. It is also to be understood that the terminology used herein to describe certain embodiments of the present invention is not to be construed as limiting.

Although many delays and methods similar or equivalent to those described herein may be used in the practice of the present invention, preferred materials and methods are described below.

As agreed above, the present invention is based on the discovery of new diagnostic methods for the diagnosis of early HCV infection. The methods are based on the identification and use of highly immunogenic HCV antibodies and antigens present in the early stages of HCV seroconversion. precision and kiss-

reducing the frequency of false results The methods can be conveniently used in this single essay format.

Specifically, the assay was performed on a solid support to which one or more HCV antigen core antibodies (directed against the same or broken HCV core epitope) were bound and an epitope from the NS3 / 4 &apos; region of the HCV polyprotein. Antibody antibodies useful in the present invention include, but are not limited to, antibody molecules, such as monoclonal antibodies, which are amino acids 10-53, 10-45, 67-88. amino acids that are directed against epitopes in the nucleus between amino acids 12013E or antibodies directed against any of the core epitopes identified in the literature (Hougbton et al., U.S. Patent No. 5,350,671 to Friene et al. Chien et al., 1993; 3. Gastroent Hepatol 3: 833-39 (1993); Chien et al., International Patent Application 94/01778; Chien, DX: WO; International Patent Publication No. 94/01778; and Serial Nos. 08 / 403,590 and 08 / 444,818, incorporated herein by reference.

The HCV polyprotein RS3 / 4a senidia has been described in the literature and the sequence and complete sequence of the protein have been described. Y & c et al; Science 7, 1353-1363 (1999).

November); Sali et al., Bioebemiahy 37, 3392-3401 (1998); Baríertsehlager, Re <1 Viral. Hepat, 6, 16S-I81 (1999);

Pasmashapatra et al., U.S. Patent No. 5,843,752 to Wash. At least one epitope from the M83 / 4a ancient expression circulating in the room

Λ Μ * _ν ··>

V νί »used in the conformation of naturally occurring HCV particles or infectious products, each of which is the persistence of protease and, if appropriate, helicase activity normally possessed by the N83 / 4a gene product and / or antigen immunoreactivity with an HCV. antibodies in a biological .mintában from an infected subject, and the epitope immunoreactivity loss of antigen denaturálásávalx the konformádóa epitope such as interruptible by heating, the pH is excessively acidic, or changing to the alkaline side, or known organic denaturálösserek such dítloíréitől (DTT) _S or corresponding detesgens by adding [Protein taíácaikm Methods, the praetíoa! approach editor; N, L, V, Harris and 8th English, IRC Press) and comparing the denatured product with the untreated product,

Protease and helicase activity can be assayed using standard enzyme assays well known in the art. Protease activity can be assayed, for example, by assays well known in the art (Takeshita et al; Analytical Biohemstry 247, 24: 2-246 (1997); Kakilebl et al., Journal of Biochemistry 122: 749-755 (1997); Sah et al. (1998) Biochemisfry 37, 3392-3401 (1999); Cho et al. (1999) J.Vlrot Meth.80, 77-84 (Powler et al. J. Blomoh Sereen). , 5, 158-158 (2000); Kim et al., (2000) Analytical Biochemistry 984, 42-48. A particularly convenient method of assaying a protease assay is described in detail in the following examples.

Similarly, helicase activity assays are well known in the art, and the activity of a BS3 / 4a eptiopic hepticase as exemplified in the wk code part! with a BUSA assay [Bioehmmeal and Rfepfeysfcel Research Communications Tjgb

¢.

*

594-599 (1993)], a scintillation assay system (Kyono et al., Analytical Biochemistry 257, 120-126 (1993)), high performance screening assays (Hicham et al., Antiviral Sl. 46, ISI-193 (2000)). Kwong et al., Methods Mot Med. 24, 97-116 (2000)] and other methods described in the literature (Khu et al., 2001, d. Virot 75, 205-214); 273: 316324 (2000); Paolini et al. (2000) J. General Virology 81, 1335-1345 (2000); Frcugsohat et al. (2000) Biochemistry 39, 5174-5183; Preugschat et al., Methods Moh Med. 364 (1998); Mosson et al. (2000) Biochemistry 39: 2619-2625.

The length of the antigen is sufficient to maintain an immunoreactive conformational epitope. The polypeptide containing the antigen used is often full-length, however, the polypeptide may be truncated, for example, to enhance solubility or increase secretion. The conformational epitope found in NS3 / 4a is expressed as a recombinant polypeptide in a cell and this polypeptide provides the epitope in the desired form as it is. will be described in detail below.

Representative amino acid sequences for H53 / 4a bp peptides are shown in Figure 3 and 4A-4D. 4A. In Figure 3, the iron scaffolds at position 182 were replaced with alanine according to the natural position at that position, in order to prevent the molecule from being self-formed. 4A-4D. The amino acid sequence depicted in Figure 2-686 in Figures 2-686 corresponds to amino acid positions 1027-171 of HCV-1. Thus, the initiator codon for methionine (Met) at position 1 is shown. In addition, at position 1428 of HCV-1 (amino acid 403 in Figure 4),

is mutated to Thr Fro and is mutated to Ser Ile under normal conditions in HCV-1 1429 (404 in Figure 4). However, in this assay both the natural sequence, N with or without a terminal methionine, the analogue described, with or without a terminal N-terminal methionine, or other analogs and fragments can be used as long as the epitope produced by this method retains or re-adopts its natural conformation, i.e., protease activity and also the activity of heukase (Dasmahapatra et al., U.S. Patent No. 5,343,752; Zhang et al.; U.S. Patent No. 5,990,276, each describing analogs of NS3 / 4a).

The N33 protease of HS3 / 4a is located approximately at positions 1027-1207 in HCV-1 numbering and in Figure 4 numbering in 2-lS2. The structure of the NS3 protease and active site is known (De Franeeseo et al., Antivir. Ther., 3, 99-109 (1998); Kocb et al., Biocheahstry 40, 631-640 (2001)). In particular, it is desirable to retain amino acid residues 1 or 2 to 155 of Figure 4 with minor or only conservative substitutions. 1A or 2-175 amino acids, most preferably 1- or 2-175 amino acids, and most preferably 1- or 2-132 amino acids. , with or without the · H-terminus metfenthal, The hsicase domain is a HCV-1-nsk powder of about 93 to 1657.

Található Φ Φ * χ φ Φ X Φ φ Φ (see position 207-632 in Figure 4). Thus, if helicase activity is required, this portion of the molecule should be retained with minimal or conservative changes. One skilled in the art can easily identify other regions that will tolerate changes in the known structure of NS3 / 4 &.

The solid support may also contain antigens. For example, multiple fusion antigens (abbreviated as &quot; MEFA *) may be bound to a solid support as described in WO 97/44469 for use in the assays in question. Such MEFAs include multiple epitopes derived from two or more of the viral regions shown in Figure 1 and Table 1. More specifically, as shown in Table 1 and Figure 1, an HCV polyprotein results in at least ten different products as a result of cleavage, in the order NHs-Core-E1-E2-p7-NS2-NS3-NS4aNS4b-NS5a-NS5b-COOH. The core polypeptide occurs at position 1191 based on HCV-1 numbering (Choo et al., 1991, Proceedings of the National Academy of Sciences, USA 38: 2451-2455, for the HCV-1 genome). This polypeptide is further processed to produce an HCV polypeptide of about 1-173 amino acids. The envelope polypeptides, E1 and E2, are located at positions 192383 and 384-746. The P7 domain is located at positions 747-809. NS2 is an integrant membrane protein with proteolytic activity and an 8 ΙΟΙ 026 polyprotein.

Nb2, in its oxime nucleus or in combination with the liver (located at positions 1027-1657), cleaves the bond between NS2-NS3, which in turn generates the N-terminal of NS3 and releases a large polyprotein, which. has both serine * protease and RNA heycase activity. The NS3 protease (located at positions 1027-1207) serves to process the remaining polyprotein. Helicase activity is found at positions 1193-1657. The completion of the maturation of the polyprotein is initiated by autocatalytic cleavage of the NS3-N34a bond catalyzed by the NS3 Serbian protease. Subsequent NS3 cleavage of the HCV polyprotein appears to involve recognition of the polyprotein cleavage sites by another subtype peptide NS3 In these reactions, NS3 releases a NS3 cofactor (NS4a, located at position 16381711), two protein (NS4b, about a

1712-1972) and NS5a, about a. 19732420) and an RNA-dependent RNA primerase (NS5b, located at positions 2421-3011).

Table I

Dome Approximate limits * C (core) 1 -191 away 192-383 B2 384-746 P7 747-809 NS2 810-1026 HS3 1027-1657 N34a 1658-1711 NS4b Í712-1972 NSSa 1973-2420 NSSb 2421-3011

ϊΐ φ φφφ χ Φ Φ * χ

X * Φ Φ Φ X X * * »<φ φφφ φφ φφ * HCV-1 numbering. See Cboo et al., 1991, Froceedings of the National Academy of Sciences USA 88: 2451-2455;

Multiple HCV antigens are part of a single, contiguous amino acid chain that does not occur in nature. Thus, the linear order of epitopes differs from the linear order in which they occur in their genome. The linear sequence of MEFA sequences generated for use here is of great interest in achieving optimal antigenicity. assembled. Preferably, the epitopes are derived from more than one HCV strain, thereby providing the ability to detect the major HCV strain in a single assay. So the. MEFAs prepared for use in the present invention contain various immunogenic regions derived from the foregoing.

In addition, in MEFAs, a protein that is a

such as Wi

It comes from 9/63 'sshiít, which is su & seges> an xsmeríí

2, 3, 4, o, or 3w-41v Ammosacids contain at least 7, 8, 9, or 10 or more of one or more epitopes derived from the HCV base protein.

epitopes derived from the hypervariable region, i.e., the 384-4e6 digestion region, may also be present in the MEFA antigen. A particularly potent E2 epitope is one that contains a consensus sequence from this region, i.e., Gly-Ser-Ala-Ala-.Arg-Thr-Ihr-Ser-Olly-Fhe-Val-Ser-Leu-Phe-Ala-. Fro-Gly-Ala-Lys-Gln-Ásn consensus sequence, which is the consensus sequence of 3904 lo antisense acids of the HCV type 1 genome. A representative E2 epitope in the MEFA of the present invention contains a hybrid epitope spanning amino acids 39-444.

* Φ 'φ X φ φ Φ * * φ φ φ φ φ φ φ φ φ * φ * φφ

Such a hybrid Ε2 epitope may contain a consensus sequence consisting of amino acids 390-460, flanked by the natural amino acid sequence of HCV E2 411-444.

In addition, antigens may be derived from different strains of HCV. Several viral strains of HCV are known and an epitope from any of the strains in a fusion protein can be used. It is well known that one individual of any organism is different from the other and that a particular organism, such as a virus, can have many different strains. For example, as explained above, HCV has at least 6 genotypes. Each of these genotypes has determinants of equivalent antigen. Specifically, each strain contains a number of antigenic determinants present in all strains of the virus, but slightly glazed with the antigenic determinant known as 5-1-1 (see Figure L). This particular antigenic determinant is present in three different strains of HCV in three different strains of virus. for this

en this taiaimany is one. 5-1-1. all three forms appear in the multiple epitope fusion antigen used in the immunoassay in question. Similarly, equivalent antigenic determinants derived from the nucleus of various HCV strains may also be present. Equivalent antigenic determinants generally show a high degree of homology in the amino acid sequence, which is homologous! » in general, 30% or more, preferably 40% or more, when the sequences are aligned. Multiple copies of the present invention may contain several identical copies of the same e. • X »

4 * 0

0 0 «00,

X * 0 0

0 0 «* X« 0 · ** 0

0 * 0 *

0 X -0Μ «0

Representative MEFAs for use in the present invention are described in WO 97/44469. Other representative MEFAs for use in the present invention include ΜΕΡΑ 12, ΜΕΡΑ 13 and ΜΕΡΑ 13.1. It will be understood that these MEFAs are representative MEFAs and that the gaseous gene from the HCV gene will also be useful in the present invention and these and other MEFAs may be incorporated.

The DNA sequence of MEFA 12 and the corresponding amino acid sequence are shown in Figures 7A-7P. 4A. The general structural formula for ΜΕΡΑ 12 is shown in Figure 6, which is as follows: hSOD-El (dpus 1) -E2 HVR consensus type-E2 HVR consensus (type 1 and 2) ~ c33c short ( Type-1) -5-l ~ l (Type-1) ~ 5 ~ 1 (Type-3) -5-1-1 (Type-2) ~ c 10) (Type-1) -NS5 (Type 1) -NS5 (Type 1) -Core (Type 1 * 2) ~ Core (Type 1 *). This multiple epitope copy contains the following amino acid sequence, numbered based on HCV1 (the numbering of amino acids shown below follows the numbering used by Cboo et al., U.S.A. 88, 2451-2455, Froceedings of the National Academy of Sciences). (1991)) (numbered as # 1 amino acid is the first methionine encoded by the coding sequence of the coding region): amino acid 1-69 of the superoside dismutase (SALT which can be used to enhance recombinant expression of the protein); amino acids 303 to 326 from the E1 region of the polyprotein: 390 to 418 amino acids of the polyprotein, representing the consensus sequence of the bipervariable region of HCV-1, aE2; amino acid residues 334-414 of the E2 region of the polyprotein, which represents the consensus sequence of the bipervariable regions of HCV1 and E2 of HCV-2; HCV-1 polyprotein 1211-1457 amiXX

Nos * nosavans, which define the helicase; three copies of epitope 5-1-1, 1689-1735-amino acids »one from HCV-1, another from HCV-3, third from HCV-2, which copies are equivalent antigenic determinants three different strains of HCV virus; HCV-1 peptide of HCV-1 C10O, amino acids 19011936 of pofiprotein; two identical copies of the epitope from the NS5 region of HCV-1, each containing the 2273-2313 amino acids of the HCV base protein; and two copies of three epitopes from the core region; two of HCV-1, and one of HCV-2, which are copies of the equivalent antigenic determinants of amino acids 9-53 and 64-83 of HCV-1 and 67-84 of HCV-2. es amino acids

Table 2 shows the amino acid pores of the 12 different epitopes of,, 7A-7P. The table is numbered according to HCV-1 {Choo et al; Proceedings of the National Academy of Sciences (USA) 38: 2451-2455 (1991). MEFAs 12 and 13.1 also contain the general formula specified in ΜΕΡΑ12 above, with modifications shown in Tables 3 and 4.

ΜΕΡΑ 12 | sweet as #

I 72-89 92-112

pel

MiWrtSVAVrt \ "

ΓΪΪ3-143

I 146-392

I390-410 Consensus

E2 HVk 142 {384-410 _i IW ~ l4S7 ~ JI ϊ

φφ * φ * «Αί« S-JS Λ · * · * *

Φ * »ΦΦΦ φ <· * Φ

X, φ Φ ΧΦ Λ ‘Φ

395-44 Ϊ SphI S-I-i 1689-1735 1 444-490 Kral 5-1-1 1689-1735 3 493-539 Clat 5-1-1 1689-1736 2 543-577 Aval Cl 00 1901-1936 1 580-615 XbaI NS5 2278-2313 .1 618-653 BgÖI N85 2278-2313 1 654-741 NcoI core epitopes 9-53, R47L 64-88 67-84 1 1 2 742-829 ............................. Ball mag 19-53, B47L 1 | § λ | 2 ........... ..................... 1 * Here's the hSOD her vein is truncated, so much so that its a detection

igatum, an Irish dye the k y used in the statement be bound to MEFA,

zett and-SOD antibody showed that anti-megakaV antibody

meia as # 5 'end location epitope hot as # breaking 1-156 NcoI mutated hSOD (70- 72 ami- nosav, Ala!) 161-178 Miul away 303-320 1 181-201 Hind! B2 HVRla consensus 390-410 1

U:

* ·?

ϊ.ϊ

202-232 E2 HVR1 * 2 consensus 334-414 1.2 235-45Ϊ C33C is short 121.1-1457 1 454-500 Hind III 5-1-1 PImut * 1639-1735 1 503-549 NrnI 5-1-1 PImuC 1639-1735 3 552-598 Cla 5-1-1 PImut * 1639-1735 2 601-636 Aval C100 1901-1936 1 639-674 XbaI NS5 2278-2313 1 677-712 BG «l NS5 2278-2313 1 713-806 core epifcopok 9-53, R47L 64-38 67-34 1 1 2 601-383 core epitopes 9-53, R47L 64-88 67-34 1 1 2

* Αζ 5-1-1 epitopes are modified, thus eliminating possible cleavage sites (CS or CA) driven by the NS3 / 4a recombinant protein. Instead of CS or CA, the sequence is changed to ΡΙ. In addition, the SOD protein is altered such that the detectable conjugate, a toner peroxidase-labeled antiSOD antibody, does not bind to MEFA. The core epitope is also amused to prevent the HCV core antibodies used in the detection from binding to MBFA.

In one assay format, the sample is combined with the solid support as described below. If the pattern ♦ X Jí φ φ * · *

HCV infected, the core antigens and the HCV antibodies on the solid support against the rotating epitopes on the solid support bind to the components of the solid support. A detectably labeled antibody is then added. The labeled anti-core antibody is directed against another epitope, not against the anti-core antibody that binds to the solid support. This anti-core antibody binds to the core antigen, which is captured by solid, anti-core antibodies on the support.

An antigen that reacts with a captured HCV antibody in a biological sample that reacts with a captured sample HCV antibody

preferably an epitope derived from the N83 region of the HCV polyprotein. This antigen binds the captured HCV antibody in the sample. Many antigens are known, including these epitopes, and including, without limitation, antigens from the c33e and the cu fusion protein. These and dk are well known in the art and are well known in the art and in the literature (Honghton et al; U.S. Patent No. 5,350,671; Chien et al .; Froceedings of the National Academy of Sciences, USA 39, 10011-10015). 1992); Chien et al., J. Gastroent. Hepatol. 8, S33-39 (1993); Chien et al., WO 93/00365; Chien, D.YU WO 94/01773; and U.S. Patent Nos. 03 / 403,590 and 03 / 444,813).

A second labeled antibody is added, which. is directed against the antigen described above. This is the antibody

X * χχ · «'Χ'Χ

X X χ ΧΧ O »Ji may be directed against any epitope on the antigen. For example, the antibody may be directed against the NS3 antigen on the antigen. Alternatively, if the antigen mentioned above is expressed in the form of a fusion protein, the second labeled antibody may be directed against the fusion partner. Additional antigen-

two and antibodies may be added, especially if the solid support contains MBFA. These assay formats are described in detail below.

A representative assay of the present invention a. Figure 2 shows the solid carrier antibody clone shown in Figure 2, designated cl13 and cl 1-7. These antibodies are directed against the epitope in the N-terminal region of the nuclear protein at amino acids 10-53, which is contained in a solid support according to the HCV polyprotein sequence, a sample of NS3 / 4 &lt; + &gt; epitope _S * ai is added to the solid support. Antibodies directed against the HCV core antigen and the NE3 / 4a epitope, which are all present in the sample, bind to the capture reagents on the solid support.

Then the HCVI is a polyprotein. Sequence numbering is carried out by addition of a Cl 1-14 horseradish peroxide (HRP) labeled anti-nucleus monoclonal antibody directed to the C-terminal region of the nucleus at positions 120-130. A fusion protein containing a sequence of the human SOD (hSODj and an epitope of the c33c region) is then added, as is a second HRP-labeled antibody directed against the SOD portion of the fusion protein. The SOD-c33c fusion protein binds anti-NS3 antibody and anti-SOD antibody hos, which

Φ * φ φ φ φ φ φφφφ

φ. φ φ φ φ

ΦΦ Φ Φ «φ

-φ ψ * * φ »φ -Φ ¥ φ

Ti ¥ ΦΛ ΦΦ binds the SOD ~ c33c fusion protein, Detection of labeling for HCV infection pathways

Another representative assay of the present invention is shown in Figure 8. The antibody assay is configureed with an antigen-antibody-antigen sandwich capture assay that uses both NS3 / 4a and MBFA 12. The solid support contains two of the above-described anti-core monoclonal antibodies, an NS3 / 4a. 4 & epitcpot, as well as a representative MEFA, MBFA 12, which contains a truncated version of human superoxide disxtase. For the assay described above, the biological sample is added to the solid support, HCV core antigen, as well as in the sample. antibodies directed against the NS3 / 4a epitope and MBFA epitopes. for capture reagents on solid supports. Two antigens are added, one reacting with them, the antigen present in the sample is bound to HS3 / 4a (the other described above is reactive to the MBFA 12 binding sample, Figure 3 shows the MBFA 12 / sample antibody complexing antigen). a fusion product between a super-disdysmufase molecule and the c22ks A47-L44W, the o22ks antigen is derived from the core region and contains the Lys10 ~ Ser99 amino acids of the polyprotein and the normally present Arg47, and Len in position 44

Ap is m, The antigen detection assay is a second HFP-labeled monoclonal anti-antigen / antibody combination assay as described above, since as HCA core antigen both our HS3 / 4a and / or core antibody are Idmutathaφ * φ φφ. φ φ «$ φ» ♦ <»♦♦.

Φ Φ φ φ φΐ χ. «X φ ** Φ Φ φφί.φ ΦΦ Φ4 essays essay 'with the same medium, in the same essay. In addition, as described above, a protein derived from a finmeshift (as described, for example, in International Patent Publication No. WO) to other HCV epitopes, such as the forerunner, 5-1-1, NS5, and the polyprotein. oh in the combination cocktail, the non-structural agent of HCV.

In order that the present invention may be more fully understood, the present invention relates to the preparation of immunizing antibodies of the present invention; the preparation of an immunoassay; and here's how

in the execution of ammunition ^

ÁjjCy

As mentioned above: production of antibodies

ztuR, the total aon. which binds to a solid (e.g., one or more antimagulant antibodies) that detect anagene / eHenogen complexes when the sample is infected with HCV. These antibodies may be polyclonal or monoclonal antibody formulations, munospecific antisera, human antibodies, or they may be hybrid or chimeric antibodies, such as humanized antibodies, altered antibodies, F (abj2 fragments, F (ab) fragments, &quot;). ^ us. ^, straight-line antibodies, dimeric or trainer antibody construct constructs, mini antibodies, or functional fragments thereof that bind the antigen in question.

Antibodies are prepared in a coat well known to those skilled in the art (see, e.g., U.S. Patent 4,011,308;

4S

4,722,890; 4,016,043, 3,876,504; U.S. Patent Nos. 3,770,380 and 4,372,745). For example, polyclonal antibodies are immunized by immunizing a suitable animal, such as a mouse, salmon, sheep or goat, with an antigen of interest. To enhance immunogenicity, the antigen may be bound to the vehicle prior to immunization. . Such carriers are well known to those skilled in the art. Preferably, the immunization is accomplished by mixing or emulsifying the antigen in saline, preferably in a complete Freund's adjuvant, and then injecting the mixture or emulsion parenterally (alternatively subcutaneously or intramuscularly). The animal is usually given a second booster injection of antigen saline 2-6 weeks later, preferably using Freund's incomplete adjuvant. The antibodies may be prepared by in vivo immunization using methods well known in the art. The polyclonal antiserum can then be recovered from the immunized animal (see U.S. Patent No. 5,350,671 to Houghton et al., For a description of anti-HCV polyclonal antibodies).

Monoclonal antibodies are generally prepared by or modified by Kohlette and Müstein (1975, Nat. 256: 495-497). Typically, a mouse or a rat is immunized as described above. However. instead of bleeding the animal for serum extraction, the spleen (and, if appropriate, a number of lymph nodes) is removed and a single-cell suspension is prepared. If necessary, the spleen cells can be screened (after removal of the aspirated adherent cells), in principle, that the cell suspension is

4§

5i «* XX '** ¢. $ *> χ <·? <·> «Κ X» 5ί ** í «>« £ · „. 4 $ ί · 'W antigen coated plate or well. B cells, which express antigen specific membrane-bound immunoglobulin, bind to the plate and do not wash off for the remainder of the suspension. The resulting B cells, or all of the dissociated spleen cells, are then fused to myeloma cells. , maid in a selective medium (such as b mn.

The resulting hybridomas are then spiked by dilution and assayed for the development of antibodies that specifically bind to the immunizing antigen (and that do not bind to the unrelated antigens), The selected monoclonal antibody, hybrids, in, ritro (e.g., tissue culture bottles or hollow fiber reactors), or in. rtoo (e.g., ascites of mice).

The preparation of various anti-HCV monoclonal antibodies has been described in Houghton et al., U.S. Patent No. 5,350,671; Chien et al., U.S. Patent No. 93/00365;

and U.S. Patent Application Serial No. 08 / 444,818; Kashiwakuma et al., U.S. Pat. No. 5,871,904].

As explained above, antibody fragments that retain their ability to recognize the antigen of interest may also be used in a rotating immunoassay. Numerous Different Antibody Fragments It is known in the art that antigen binding hemeemaz are capable of exhibiting the s * binding properties of an intact antibody molecule. For example, functional antibody fragments can be generated by deleting a constant region that is not responsible for antigen binding, for example, by pepsdn excision of the antibody molecule to produce the P (ab'h) fragments. These fragments contain two antigen binding sites but lack the same. constant regions of certain heavy chains Similarly, if necessary, fragments of the Pab containing a single antigenic binding site can be produced, for example, by papain digestion of polyclonal or monoclonal antibodies. Functional fragments containing only the variable regions of the heavy chains and light chains may be prepared by standard techniques such as recombinant production

Also known as F _v (see Inhar et al., Froceedings et al., National Academy of Sciences.

Hochmsn et al., 2706-2710 (19761, Ehrlich et al.,

A single chain Fv UsPrt or "sePv *) polypeptide is a covalently linked Vh-V1 heterodimer that expresses a gene fusion that contains a gene encoding Va and Vt linked by a peptide encoding peptide (Muston et al., Proceedings). 85: 58795883 (1988)] A number of mediums have been described that allow naturally occurring aggregation of a V antibody region, but to detect and develop chemically separated light and heavy chain polypeptide chains. sFV molecule, which is wound into a harem dimension, similar to that of the antigen binding site (see, e.g., 5,091,51.3, 5,132,405

A S A. ** »* **,

U.S. Patent No. 4,946,773). The sFv molecule can be described using methods published in the art (Muston et al., 1968, Proceedings of the National Academy of Sciences, VSA 35, 5879-5833; U.S. Patent Nos. 5,091,513; 5,132,405; and 4,946,778. The design criteria include the definition of a stroke that covers the C-terminus of one chain and the. the liner between the N-terminus of another chain is generally a small hydrophilic amino acid group not prone to form a envelope or secondary structure. These methods have been published in the literature (see, for example, U.S. Patent Nos. 5,091,513, 5,132,405 and 4,946,778 to Amber Bubbles). Corresponding linkers generally consist of alternating glycine and serine residues, and may include glutamic acid and lysine residues inserted therein to enhance solubility.

"Substances * or pn bodies" can also be used-

chains containing at their C-terminus oligomeric domains which are separated from sFv by a wrist-sen (Pack et al., Biochemisfry 31: 1579-1584 (1992)). The ohgomerization domain contains self-associating α-helices, such as lencin zippers, which can be further stabilized by the addition of further disuhid bridges. The oligomerization domains are designed to be compatible with vectorial membrane winding, a process that facilitates the intrinsic winding of pelipepid into a functional binding protein. Miniatures are generally prepared by recorobinant methods well known in the art [Pack et al.

Biochemistry 31, 1579-1584 (1992); Cumber et al., 1992, J. of Immunok 149B, 120-126.

Preparation of Antigens for Use in the HCV Immune Assay As explained above, the molecules of the present invention are generally prepared by recombinant methods, so that polynucleotides encoding the HCV antigens to be used in the present invention can be prepared using standard techniques in molecular biology. For example, the above-described polynucleotide sequences encoding molecules may be obtained by recombinant techniques, such as screening of cDNA and genomic libraries, from cells expressing the gene, or from a vector known to contain the gene. In addition, the desired gene may be directly isolated from viral nucleic acid molecules using techniques known in the art (Houghton et al., U.S. Patent No. 5,350,671). The gene of interest may be synthesized instead of being cloned. The complete sequence is constructed from overlapping oligonucleotides prepared by standard methods and then a complete coding sequence is prepared (Edge: Naturre 292: 756 (1981); Nambair et al., Science 223: 1299 (1984); J'ay). et al., Journal of Biological Chemistry 259, 6311 (1984).

Thus, a particular nucleotide sequence may be prepared from vectors carrying the desired sequences, or may be synthesized, in whole or in part, using various synthetic techniques known in the art, e.g.

Φ φ «Φ> ♦ X

X * Φ> <X * φ ν φ φ φ φ φ φ φ φ

Φ Φ X φ φ Φ φ X φ

Mu Φ X Φ * Φ φ Φ φ φ φ φ φ mu mu mu mu mu mu mu mu φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ X φ specific muffagenesis and polymerase chain reaction (POR) technique, when appropriate (Sambrook et al., Molecular Cloning: A Laboratory Manual; 2nd edition, a method of aligning a complementary set of overlapping oligonucleotides produced in a conventional automated pclmucleotide synthesizer, followed by ligation with an appropriate DNA backbone, and amplification of the ligated nucleotide sequence by polymerase chain reaction, payaraman et al; 83: 40844088 (1991)). In addition, the oligonucleotide-driven synthesis according to the invention (Jones et al., Naturre 54: 75-82 (1986)), an existing oligonucleotide-directed mutagenesis (Riechmann et al., Naturre 332, 323-327 (1988); Verhoeyen et al. Science 239, 1534-1536 (1988) 1 and the word ohgc

enzymatic filling of srazza vaxo by IQueen et al., Proceedings of t

&Amp; of Sciences, USA 36, 10029-10033 (1989)) can be used to produce cells that have altered antigen binding properties and / or reduced immunogenicity.

Once the coding sequences have been prepared, or .k, these sequences can be cloned into an appropriate vector. Many cloning vectors are known to those skilled in the art and it is up to the skilled artisan to select the appropriate cloning vector. Suitable vectors include, but are not limited to, plasmids, phages, transposons, cosmids, chromosomes, or viruses, which are capable of replication when bound to appropriate control elements.

The coding sequence is then placed under the control of appropriate control sequences, depending on the system used for expression. Thus, the coding sequence is placed under the control of a promoter, a rhizomal binding site (for bacterial expression) and optionally linked to an operator by transcribing the DNA sequence of interest into RNA in a suitable transformant. The coding sequence either contains a signal peptide or or not, which is then removed by the host in post-translational processes (see, for example, U.S. Patent 4,431,739; 4,425,437; U.S. Patent 4,338,397).

In addition to the control sequences, it may be desirable to add regulatory sequences that allow expression of the sequences relative to the expression of the host cell. Regulatory sequences are known to those skilled in the art, and include those that cause expression of the gene to be turned on or off in response to chemical or physical stimuli, including the presence of a regulatory compound. Other types of control elements may be present in the vector. For example, enhancers may also be used to enhance expression levels of constructs. Examples include the early (caused by Dijkema et al; 1985; EMBO Journal 4, 761 (1985)) enhancer / promoter of the Rous sarcoma virus (LTR) gene (Gorman et al., Proceedings of the National Academy of Sciences). , USA 79, 6777 (1982)), such as those derived from the human cytomegalovirus pBoshart et al;

X ·>

XX · X * φ <X «* x x ** ** (1985)], that is, elements of the intron sequence of the dtomegalovirus A (U.S. Patent No. 5,688,688). The expression cassette may further comprise an origin of replication for an autonomous replication in a suitable host cell, one or more selectable markers, one or more restriction sites, high copy number ability, and a strong promoter.

An expression vector is constructed such that the coding sequence in question is located in the vector along with the corresponding regulatory sequences, and the position and orientation of the coding sequence relative to the control sequences is such that the coding sequence is transcribed under the control of the control sequences (i.e., DNA). RNA polymerase fused to control sequences transcribes the coding sequence). Modification of the coding sequences of the molecule of interest may be desirable to achieve this goal. For example, in some cases, it may be necessary to modify the sequence to align with the control sequences in the proper orientation, i.e., to maintain the reading frame. The control sequences and other regulatory sequences may be linked to the coding sequence prior to insertion into the vector. Alternatively, the coding sequence may be cloned directly into an expression vector that already contains the coding sequences and an appropriate restriction site.

As explained above, it may also be desirable to produce the antigen, mutants or analogs of interest to us. This is particularly true for NS3 / 4a. Methods for accomplishing this have already been published in the literature [Pasmahapatra et al., U.S. Pat. No. 5,843,752 * * * φ φ * ♦ Φ φ X Φ X ΧΦ φ

X X Φ Φ ** ΦΦΦ «Χ.ΦΦΦ Φ Φ φ φ

Φ * * χ Φ Φ Φ Φφ Φ X

United States Patent Hefi Patent; Zhang et al .; U.S. Patent No. 5,990,276 to U.S. Pat. No. 5,990,276.) Mutants or analogs of this and other HCV proteins prepared for use in the assays in question may be prepared by deleting a portion of the polypeptide coding sequence of interest, inserting a sequence, and / or by replacing multiple nucleotides. For a person skilled in the art a. techniques for modifying nucleotide sequences, such as site-specific mutagenesis, are well known (Ssmhrook et al., Molecular Cloning; The Laboratory Manna; 2nd Edition, Cold Spring Karbor Press, Cold Spring Harbor, 1989; Kunkel, TA "; Proeeedingo"). 82: 448 (1985); Gersselsöder et al., BioTechniques 5: 786 (1987); Zeller and Smith: Methods in Enzymology 100: 468 (1983); Dalbie McFarland et al., Proeeedings of the National. Academy of Sciences, VSA 79, 6409 (1982)].

The molecules can be expressed in a variety of systems, including insect, mammalian, bacterial, vital and yeast expression systems as is well known in the art.

For example, insect cell expression systems, such as baculovirus systems, are known to those of skill in the art and are published in the literature (Summers and Smith; Texas Agricultural Experiment Station Bulletin No. 1555 (1987)). Similarly, bacterial and mammalian cell expression systems are known in the art and published in the literature by fSambrook et al., Molecular Cloning: A Laboratory Manual; 2nd Edition, Cold Spring Hatbor Press, Cold Spring Harbor, N.V. Yeast expression systems are also known in the art57 and published in the literature (Theory, Genetic Engineering, Bárten et al., Buttenvorths, London, 1989).

Many suitable host cells are known for use in the above system. For example, mammalian cell lines are known in the art and include guarded life. cell physician Collection (ATCCl k available from Amencan L. such as Golden Hamster Ovary Cell (CHO), HeLa Cell, Baby Hamster Kidney (SHK), Monkey Kidney Cell (COS), Human Embryonic Kidney, human hepatocellular squamous oocytes (such as Hep G2), Madin-Barby bovine kidney cells and other cells. Similarly, bacterial hosts such as Bsekarichia eoS, Baed & m sahhks, and tkmptoeneeas spp may also be used, The yeast hosts useful in the present invention include Saoehurompees eerems & xe, Candida olbicons, Candia makoso, Jfonsemda jxdgvnotphn, Mupwmmpees / fupais, Aint / rnroetondes, , Sctozosaochömmpoes pomhe and Farrowin aphoh / dea> Insect cells suitable for use in baculovirus expression vectors include aedes peptiti, Aufographa ea ^ forniea, Bomfegx mór, Ikosophda mnlanopasfer, Apodopfem jtugipenáa and BteAcphmia n £

Nucleic acid molecules containing nucleic acid sequences of interest may be stably integrated into the genome of a host cell, or may be stably maintained on a stable episomal element in a suitable host cell known in the art.

Φ φ χ φφφ φ Φ φ

Φ Φ.Φ φ X * ΦΦΦ

Φ Φ Φ X Φ Φ Φ Φ Φ using ion-gen gen-b .ummhi techniques (see Baker, U.S. Patent No. 5,399,346).

Depending on the expression system and host selected, the molecules are produced by growing host cells transformed with an expression vector as described above under the conditions under which the protein is expressed. The expressed protein is then isolated from the host cells and purified. If the expression system secretes the protein into a growth medium, the product can be purified directly from the medium. If not secreted, it can be purified directly from cell lysates. Appropriate propagation conditions and recovery methods are within the skill of the art.

The preparation of various HCV antigens by recombinant techniques has been published in the literature (Houghton et al., U.S. Patent No. 5,350,671; Chien et al., J. Gastroent. Hepatol. 3, S33-39 (1993); Chien et al., 94). International Patent Publication No. 01778; Chien, DY: WO 94/01773.

New Magnesium Assays Once prepared, the above materials and HS3 / 4a antigens are transferred to a suitable solid support for use in the immunoassay in question. As a solid carrier, for the purposes of the present invention, it may be any material which is an insoluble matrix and may have a rigid or semi-rigid surface. Solid carriers include, but are not limited to, substrates,

5§ φ * ♦

Φ · Φ ‘Φ <-χ · φ φ. φ φ> φ

Φ φ φ Φ Φ ν

Φ Φ- Φ χ

Φ Φ Φ * Χ φ φ such as nitro-cellulose (membrane or microtiter well); polyvinyl chloride (such as plates or microtiter wells); polyazyrene latex (e.g., beads or microtiter plates); polyviolide fluoride; diazotized paper; nylon membranes; activated beads; magnetically reactive beads and the like. Such carriers include plates, grains, disks, capillaries, hollow fibers, squash, solid fibers, cellulose beads, porous glass beads, silicone gels, polystyrene beads, optionally cross-linked with divmylbenzene, grafted copoly ring, , with cross-linking with latex stoichloroylenediamine, and glass fragments coated with a hydrophobic polymer.

If necessary, the molecules to be bonded to the solid support can be easily functionalized to be resin or acrylate. units, allowing molecules to be incorporated into other polymers, e.g.

atoa, rn tm.Oazo.iOa, polythiophenic, polyether, sichelic gel, azoane, polyphosphate (edge, asarose, cellulose and the like).

responds to HCV and core eUeners and NS3 / 4 &

(hereinafter collectively referred to as the "solid phase *") and optionally with one or more MBFAs, under existing conditions that allow the molecules to immobilize properly to the support. Occasionally, immobilization on a carrier can be inhabited when anti * * χ · ·> first

«· *« Xx · χ * ❖ ί ¥ «*> <· <·>

* *. $ «♦>« «*><>> Α <* X _s v gene and / or antibody is coupled to a protein having better solid phase-stone properties. Suitable linking proteins include, but are not limited to, macromolecules such as serum albumin, including bovine serum albumin (BSA), sponge hemodanin, immunoglobulin molecules, thyroglobulin, and evalbumin. , proteins well known to those skilled in the art. Other reagents used to attach the molecules to the carrier include, for example, polysaccharides, lactic acid, oleic acid, polymeric amino acids, amino acid copolymers, and the like. Molecular methods for binding these antigens are well known to those skilled in the art (Brinkley, MA: Bloconjugate Chem. 1992, 3, 2-13;

Hashida et al., J. Appl. Biochem. 6, 56-63 (1934); Anjaneyulu and Stores; International Journal of Protein and Peptide Research 30, 117-124 (1987)).

After reacting the solid support with the solid phase components, washing will remove all non-munobitalized solid phase components from the support and contact the support components with the biological sample under appropriate binding conditions, which are presumed to contain HCV antibodies and antigens. collectively referred to as Jigandum molecules *). After washing to remove all unbound ligand molecules, a second anti-core antibody is added to the system, which, under appropriate binding conditions, is directed against another epitope, not the antibody bound to the support. The added anti-core antigen contains a pond ski * Φ ¢. Φ The fcw.

• * Φ * * A Φ $ # A A A A * AA * «ΦΦ χ. χ. *, φ * Α X * Α .φ ΑΑ The Α can be labeled as described above and appears to bind to any core antigen that may be present in the sample that has reacted with the carrier-bound anti-core antibody. In addition, one or more antigens are added to the system which are capable of reacting with antibodies in the sample, which in turn react with the NS3 / 4a epitope. As explained above, the HCV polyprotein is typically derived from the N83 slice, most of which is from the HCV c33c region (Houghton et al., U.S. Patent No. 5,350,671; Chien et al., Froceedmgs of the National Academy of Sciences, USA 89). 1001.1-100X5 (1992); Chien et al., International Patent Publication No. 93/00365; and U.S. Patent Nos. 08 / 403,590 and 08 / 444,818, describing this region and epitopes derived therefrom) . A labeled antibody directed against this antigen is also added. The antibody therefore binds the antigen, which reacted with the anti-NSS antibodies in the sample. For this purpose, e33c can be provided in a conventional manner by fusion between c33e and human sdMD (recombinant methods) (see, e.g., Honghton et al;

U.S. Patent No. 5,350,671 to Allam et al. The nucleotide sequence of the human superomod dismutase is known (see, U.S. Patent No. 5,710,033 to Hallewell et al.). The labeled antibody to human superomod dismutase can thus be used to detect the epitope NS3 / 4a, e.g. complexes formed in the sample reacting with this epitope and HCV polypeptides which in turn bind the antibody in the sample.

If a ΜΕΡΑ is on a solid support, one or more additional antigens that react with the antibodies in the biological sample that are bound to the antigens on the MEFA can also be added to the assay. An antigen from the HCV core region, more particularly the e22 antigen, is particularly useful in this regard. contains 119 N-terminal core amino acids of the HCV polyprotein One antigen from c22 is the c22ks A47-L44W, which contains the Lyslo-Ser99 amino acids of the polyprotein, and Arg47, which is present under normal conditions, is excised and Trp instead of flax. It is. As for the c33c epitope described above, this antigen can be provided in the form of a hSOD fusion, and the same labeled antibody to human supereroadd dismutase can be used to detect the antibodies in the sample and the complex formed between the epitope NS3 / 4a and / or amely complexes also bind to HCV antigens (e.g., o33c and e22).

More specifically, a BUSA method can be used in which wells of a microtiter plate are coated with the solid phase component. A biological sample is then added to the coated Incas containing, or presumably containing, Ugandam molecules. After incubation time sufficient for the hgandum molecules to bind to the immobilized solid support, the plate (s) may be washed to remove the stencil units and a detectably labeled secondary antibody molecule (labeled antimicrobial antibody) and NS3. an epitope-containing molecule and an antibody against the epitope containing the N83 epitope is added to rendazer. Allow these molecules to react with any of the capturing antigens and antibodies, wash the plate, and detect the labeled antibodies using methods well known in the art.

The above-described assay reagents, including the immunoassay solid support with bound antibodies and antigens, as well as antibodies and antigens that react with the captured sample, can be provided in kits with appropriate instructions and other necessary reagents for the purpose of performed. The kit may also contain appropriate labels and other packaged reagents and materials (such as wash buffers and the like), depending on the immunoassay selected. The standard inxmnnesses, such as those described above, can be executed using these kits.

fiLJÖiérfetireaz

As a matter of fact, the leien mus am: many monographs are ae sak any illustration

. «Get specm examples. By way of example and not stem, the accuracy of the present invention (amounts, temperatures, etc.) should be assured, but the price is accurate. defects: and differences of course front immune? / elier

The HCV antigen / antibody combination immunoassay of the present invention is compared with other HCV assays to limit the detection limits of azero & inversion, and these are determined by $ * V Φ * * ·>

** * ¥ * ¥ * φ $ ¥ ¥ Φ Φ ** ΦΦφ.

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The “Materials and Methods

blood samples

Panels of human blood samples from commercial jitters were used. Such panels are available, for example, from Boston Biomedica, Inc. of West Bridgewater, MA (BB1); Bioclinical Fartners, Prarddin, MA (BCF); and North American Biologics, Inc. » BoeoRatan, FL (NABl). 5 and he. days shown in table are the days on which the blood from k soupf ”^ ^s '

Mpn.okionáils.ellenanyagek

The monoclonal antibodies cl 1-3, cl 1-7 and cl 1-14 are as follows

Ckraeál nntan, New Jersey) we got them.

Antibodies cl 1-3 and cl 1-7 are directed against the N-terminal portion of the nucleus (amino acids 10-53 of the HCV1 pali protein numbering). Monoclonal antibody cl 1-4 is directed against the O-terminus of the nucleus (amino acids 120-130 of the HCV1 polyprotein numbering). Antibodies 1-4 are conjugated to horse radish gas by standard procedures.

Monoclonal antibody 5A-3 is an anti-superoside dismutase antibody directed against amino acids 1-65 of s.eoperoid dim'nnW and can be prepared by standard techniques. The antibody is conjugated to horseradish peroradiolase on a mat described below.

ίί - «ΧΦ õ5

The c33c antigen (266 amino acids, amino acid 11921457 of the HCV polyprotein) is expressed as an internal superoxide dismutase fusion protein using the methods described for the synthesis of 5-1-1 antigen in Eschericho colo (Choo et al., Science 244: 359-362 (1989)). Purification of the recombinant antigen was reported by Chien et al., 1989, Chien et al., 1989, 89, 10011-10015. See SOD-c33c Production Protocol; Houghton et al., U.S. Patent No. 5,350,671.

The NS3 / 4a epitope used in the assay is a conformational epitope having the sequence shown in Figure 3.

C.. Immune Forms

The Abbott PRISM assay (Abbott Laboratories, Abbott Park, l'L) is commercially available and is an antibody-based detection assay. The essay was done according to the manufacturer's instructions,

The ORTHO HCV Version 3.0 ELISA Test System (hereinafter referred to as the Ortho 3.0 assay, Ortho Clinal Diagnostics, Raritan, New Jersey) is an antibody-based detection assay. The assay is performed according to the manufacturer's instructions,

Roehe Amplicor Assay (Roche, Pleasant, CA) is a commercially available polymerase chain reaction assay. The essay a. according to the manufacturer's instructions,

The Gen-Probe TMA assay (San Diego, CA) is a commercially available transcriptional amphiphication assay. The assay is performed according to the manufacturer's instructions.

Ortho Antigen Assay (Ortho Clinical Diagnostics, Raritan,

Jersey) is an antigen-based detection assay. The assay is performed according to the manufacturer's instructions,

The HCV antigen / antibody combination immunoassay in question is performed as follows, 4-4 mg / l of purified monoclonal antibodies C1-17 and C1-13 (Ix phosphate buffered saline, pH ~ 7.4). and mix thoroughly. The recombinant NS3 / 4a antigen is added to the same coating buffer. The solution was stirred for 30 minutes before coating. 200 μΙ of the above solution was added to each 96-well Costar medium binding microtiter plates (Corning, Inc.). The plates were incubated at 15-30 ^o C for 16-24 hours. Plates were washed twice with distilled water, then with 300 µl / well post-buffer (1% bovine serum albums (HSA), 1x PBS) for 1 hour, then 300 μΐ / well stability buffer (IxPBS, 1% BSA, mannate, polyethylene glycol). (FEG), gelatin) for 1 hour. The plates are aspirated and dried for 24 hours at 4 ° C in a lyophilizer. Plates are placed in a bag with desiccant.

To perform the antigen / antibody combination bioassay, 100 μΐ of amplified lysis buffer (1% N-lauroyl sarcosine, 0.6 M sodium chloride, 50 mg / ml mouse IgG (technical grade, Sigma, St)) was added to the plate. , bouis, MO), B'SA (Bayer) modified with 1% sulfhide ™, 0.1% casein). Then 100 μΐ of sample is added. Shake for one hour at 40 ° C. Plates were washed six times with IxFBS, Tween-20, in an Ortho plate washer. 200 μ! conjugate solution (1:75 dilution of cl 1-14-HRF 250 ng / assay SOD-c33 antigen plus 1: 5000 dilution in mouse anti-SOD-HRP HCV 3.0 sample diluent (ORTHO HCV Version 3.0 BUSA Test System, Ortho Clinical Diagnostics, Rarítan, »* X

New Jersey), SALT extract, each prepared 30 minutes before addition). The solution is incubated for 45 minutes at 40 ° C with shaking. This was washed six times, and the xx bag (described above) (2 ml ml tablets / ml) was added. The ÖDF tablet contains o-Phenylenediamine dihydrochloride and Hydrogen Peroted for the hormone perturbation reaction, available from Sigma (Sigma, St. bouis, MO), incubated for 30 minutes at 15-30 ° C in the dark, The reaction was quenched by the addition of 50 mL of 2M H2SO4, and the plates were read at 492 nm for 690 nm absorbance as a control.

D, Results

The results of the different assays are shown in Table 5 and Table 6 for two separate HCV-infected blood samples as described. Shaded areas indicate the detection of the virus. As described below, Chiron showed seroconversion in the antigen / gonadal assay in each sample, whereas all other antibody and antigen-based assays failed to detect seroconversion in at least one sample. Specifically, none of the antibody-based assays showed seroconversion until at least day 18 (Table 5). Table 6 shows that none of the antibody-based assays showed the presence of HCV infection by day 22. In addition, Ortho's antigen-based assay failed to detect seroconversion from day 85 onwards.

Thus, it is clear from the above results that the new combination antibody / antigen assay reduces the number of false negative results obtained with other conventional antibody and antigen based assays.

5, Table

HCV seroconversion

Sun Abbott PRISM Ortho 3.0 Roche Amplicor Gene Pro introduced TMA Orth Branch Chiron Ag / Ab 0 04 0.0 BLD 041 0.5 13 b, l 0.0 > 5x10 " 44.0 3.0 ··; 20 04 0.0 > Sxlö " 24.2  14th . 22 0.3 0.0 > 5x10 " .29,2 4 6 4 85 5 _f 44 44 '5 BQE 0.06 / 1.1 4 131 4.3 444 BQR 0.09 1.0 135 44 4.7 3x10 ». 0.09 42 i 138 5 _s S 44 BLD 0.08 14 146 5.9 4.7 BLD 041 4 24

φ * φ

φ

Φ * κ

BQR

1 »

M ~ S§3Z4aUmniom ^^ Thanksgiving Cycle

A conformational epifope of NS3 / 4 & This epitope has a. 4A-4D. which provides the S 'Hindin cloning site from the natural sequence, followed by the codons for the ACAAAACAAA sequence, the ATG initiator and HCV1a, which starts at X277a and continues to the Bgu site at amino acid 1046; b) a 638 bar pair BgO-Clal restriction fragment (encoding amino acids 1046-1274) from pAcHLTns3na4aFI; and ej a pSP72 vector (Promega, Madison, WI, GenBank / BMBL accession number X6S832) which is Hindi! and Clal are digested with a restriction endome and gel purified. Purified pAcHLTns3ns4aP1 plasmids are derived from pAcHLT, a baculovirus expression vector commercially available from BI3 Pharmingen (San Öiego, CA). Specifically, a pÁcHLT .EcoR1-Psti vector was constructed as well as the following fragments: EcoRI-Alwnl, 935 bp, corresponding to amino acid 1027-1366 of the HCV-1 genome; Alwn1SacH, 247 bar pairs, corresponding to the 1336-14X9-00 anrino acids of the HCV-1 genome; BmfhBgll, 175 base pairs, corresponding to amino acids X449-159 -9 of the HCV-1 genome; BglII-PztI, 619 bp, corresponding to amino acids 1510-1711 of the HCV-1 genome plus the transcriptional terminal codon. A SaeH--.Hm.il contains almost 70 fragments of 91 bp, corresponding to amino acids 1420 to 1448 of the HCV-1 genome, containing the P1 mutations (mutated to Thr-1428 Pro, Ser-1429 Ile). ligation with the 175 bp Hinff-BglII fragment) and then ligated to the 619 bp described above and subcloned into a pGEM ~ 5Zf (+) vector digested with SacI and PstI. PGEM-5Zf (4j vector is a commercial Escheric / ua coK vector (Promega, Madison, WJ, GenBank / EMBL accession no. X65308). After transformation of competent HB101 cells, each clone is subjected to miniscreen analysis and its sequence verified. a 885 bp Sacl-PstI fragment of pGEM5.Pl clone2 was gel purified, ligated with the 935 bp EcoRl-Alwnl fragment, the 247 bp Alwrd-Saell fragment, and the pAeHLT EcoRl obtained with the pAeHLT described above. construct name pAcHLTns3ns4aPI.

The ligation mixture described above was transformed into competent HB100 cells and plated on Luria a.gar containing 100 pg / ml ampicillin. Analysis of each clone by miruprep could identify putative positive clones, two of which were amplified, plasmid pSP72 laHC DNA, clones # 1 and # 2 were generated with a Quiagen Maxiprep kit and sequenced.

Then, the following are ligated: a) A 761 bp HindIII-ClaI fragment from pSP72aHC # 1 (pSP72.1aHC is prepared by ligating HindIII and Clal restriction enzymes with pSP72, synthetic oligonucleotides). , which secures the 5 HindIII cloning sites, is followed by the ACAAAACAAA sequence, the ATG initiation codon, and the HCV la codons, which start at amino acid 1027 and continue to the BgH site at amino acid 1046, with a size of 638 bp. -ClaI restriction fragment (coding for amino acids 1046-1274) from pAcHLTns3ns4aPI b) a 1353 base pair BamHI-HindIII fragment for the ADH2 / GAPDH yeast hybrid promoter; c) a 1320 base pair Clal-SalI fragment (encoding amino acids 1046-1711 of HCV 1a, mutated to Thr Pro 1428 and Ser Ile 1429) from pAcHLTns3ns4aPI; and d) the yeast expression vector pBS24.1, digested with BamHI and SalI, dephosphorylated and gel purified. The ligation mixture was transformed into competent HB101 cells and plated on Luria agar plates containing 100 μg / ml ampicillin. Miniprep analysis of each clone led to the identification of clones containing the expected 3446 bp BamHISall insert containing the ADH2 / GAPDH promoter, the initiator ATG codon, and HCV la N'S3 / 4a 1027-1711. amino acids (shown in Figures 4A-4D as amino acids 1-686) in which Thr 1428 (40A in Figure 4A-4D) is mutated to Pro and Ser 1429 (4A-4D in Figure 4A-4D). In Figures -4D, it is mutated to 404 amino acid Ile. The construct is designated pd.HCVla.ns3ns4aPI (see Figure 5).

The Sncchnrorm / ees oererisme AD3 strain was transformed with the pd.HCVla.ns3ns4aPI construct and the expression of some transformants was checked after depletion of glucose from the medium. Recombinant protein is highly expressed in yeast, detected by Coomassie blue staining and *> X ** «φ Φ

Κ «*

X φ φ φ «Φ ν χ immunoassay was confirmed using a polyclonal antibody against the helicase domain of NSS.

^ LMÉg

The conformational epitope NS3 / 4a was purified as follows. Soochorompees cells expressing the NS3 / 4a epitope are harvested as described above. Cells Kzts suspended in buffer (50 mmol / 1 RIS pH ~ 8, _f Ö 1:50 mmol / 1 NaCl, 1 mmol / 1 BDTA, 1 mmol / 1 fenibnetilszulfonibluorid, 0.1 pmol / l pepstatm, 1 pmol / l leupeptin) and Ein Dyno-Mül (Wab Willy A. Bacholon, Basel, Switzerland) or an equivalent apparatus Imitated using glass beads using a 1: 1: 1 cell to buffer:? 5mm glass bead ratio. The lysate 3ülÖ0xg with centríüigáljuk 30 minutes at 4 ^o C, maid added to the pellet containing the insoluble protein fraction a mosópufiferhez (6 ml / g khnduláei cell pellet weight) and shaken for 15 minutes at room temperature. Wash Buffer Composition 50 mM NaPO4 pH * 8.0, 0.3M Sodium Idoride, 5 mM β-mercaptoethanol, 10% Glycerol, 0.05% Eclucosol, 1 mM BDTA, mmol / l phenylmethylsulfate, 0.1 pmol / l pepstaftn, 1 μηιοί / ΐ leupeptin. The cell debris was removed by centrifugation (50 ° C, 30 rd, 4 ° C).

The protein from the pellet is extracted as follows. 6 mg / ml extraction buffer was added and the mixture was shaken for 15 minutes at room temperature. Composition of extraction profile: 50 mM TBIS β, β 1.0 M sodium chloride, 5 mM β-mercaptoethanol, 10% glycerol, 1 mM, 1 mM · fenilmetilszuifonil

Φ Φ S * * φ * φ ΦΦ

fluoride, 0.1 μηιοΙ / l pepstatin, 1 pmol / l leupeptin, Supernatant is maintained and added to 17.5% ammonium sulfate using the following formula: volume of supernatant (ml) multiplied by x % of / fl-x% ammonium sulfate) ^aa ml 4.1 mol / 1 with saturated ammonium sulfate, which is added to the supernatant, ammonium sulfate was added dropwise with stirring on ice, and the solution was stirred for 10 minutes on ice, The solution is centrifuged at 170000 g for 30 minutes at 4 ° C and the pellet is stored and stored at 2-8 ° C for up to 48 hours.

The pellet was resuspended and run on a Poly U column (Poly U Sepharose 48, Amersham Pharmacia) at 4 ° C as follows. The pellet is resuspended in 6 ml / g of pellet in Polv U equilibration buffer. The equilibration buffer contains 25 mM I-IEPES pH 8.0, 280 nM sodium chloride, 5 mM dithiothreitol (freshly added), 10% glycerol, 1.2 octyl glucoside. The solution is shaken for 15 minutes at 4 ° C and centrifuged at 3100 x g for 30 minutes at 4 ° C.

A Poly U column (1 ml resin per gram of starting pellet) was prepared. The linear flow rate is 60 cm / h and the charge flow rate is 133% of 60 cm / h. The column is equilibrated with equilibrating buffer and the resuspended ammonium sulfate pellet, supernatant, is loaded onto the column. Wash the column with equilibration buffer until the baseline is reached and elute the protein stepwise with Polv V elution buffer: 25 mM HEPES pH 8.0, 1 M sodium chloride, 5 mM dithiothreitol (freshly added), 10% glycerol, 1.2 octyl glucosol. The column eluate was run on SDS-polyhacrylamide gel (stained with Coomassie <· * · $ · ** * ♦ * *) and stored at -80 ° C. The presence of NS3 / 4 & epitope was confirmed by Western blotting using a polyclonal antibody against the NS3 protease domain and a monoclonal antibody (epitope 5-1-1) (HCV a).

In addition, protease activity during purification. as follows. A sample containing the HS4a peptide (KKGSVVXVGR1VWGKFAHFKK) and the N53 / 4 &apos; conformation epitope was diluted in 90 μl reaction buffer (25 mM TRIS pH **, 5, 0.15 M NaCl, 0.5 mM EDTA, 10% glycerol, 0.05 n-dodecyl B'D maltoside, 5 mM dithiothreitol) and then allowed to stir at room temperature for 30 minutes. 90 µl of the mixture (Costar, Inc., Corning ', NY) and 10 µl of HCV substrate (AnaSpec, Inc., San dose, CA) were added to the microtiter plate. The plate was mixed and spiked with a Ftuostar dilution salt. (RFU),

Using these methods, the 1 M sodium chloride extraction solvent contained 3.7 RFU / min activity, the ammonium sulfate precipitation activity was 7.5 .RU / min, and the activity of the U purification product was 13.5 RFU / min. volt.

The following competition assay is performed with the purpose of conforming epitope NS3 / 4a

major antibodies. M. HS3 / 4a antigen with c200 antigen is combined as follows.

0.5 pg and 1.0 pg NS3 / 4a prepared as described above, or c200 (Hepatology 15, 19-25 (1992)) are available from the ORTHO HCV '3.0 ELISA Test System (Ortho-Clinical Diagnostics, Raritan, New Jersey)] with 20 μΐ PHV914-5 sample (an early seroconversion blood obtained from the blood of an infected individual) in a total volume of 220 μΐ (1 * PBS). Incubate in microtiter wells at ⁰ C. The mixture is then transferred to wells coated with NS3 / 4a and incubated for 1 hour at room temperature, and the plates are washed and assayed as follows.

pg c200 antigen was added to 10 μΐ of PHV914-5 in a total volume of approximately 220 μΐ. The mixture is incubated for 1 hour at 37 ° C in a microtiter well, then 200 µΐ is transferred to an N83 / 4a-coated plate (100 ng / assay) and incubated for 1 hour at 37 ° C. Plates were washed five times with 1 * PBS, 0.1% Tween-20. The conjugate described above. 200 μΐ of the solution was added, and the plates were incubated and analyzed, Controls which were PHV915. and l * PBS (without antigen) are also treated as described above.

The percent inhibition shown in column 4 is shown in Table 7. results are calculated using the formula 3, column minus (column 2 divided by column 3, multiplied by 100). As can be seen, the data show that NS3 / 4a is neutralized by early seroconversion antibodies whereas c200 is not. A strong signal was obtained when the antibodies in the PHV914-5 c33e early seroconversion panel react with the NS34a on the plate. coating. The c200 antigen is not neutralized by these antibodies. This is shown in the upper panel of Table 7. When NS34a is mixed with a

PHV914-5, then it is neutralized and therefore there is no antibody in the sample to react with NS34 &lt; RTI ID = 0.0 &gt; Coating &lt; / RTI &gt; The data show that N334a shows another class of antibodies ^ not what c2Ö <X

Table 7

Competitive assays to detect that the NS34a antigen exhibits a different class of antibodies than the c20O antigen,

1 2 3 4 c200 v PHV914-5 control X * PBS Inhibition 50 the s i kg 1,450 1.645 12 1 pg 1.545 1,687 S 0> 5PG 1,557 1.913 19 0.5 pg 1,719 1,804 5 NS3 / 4a 4 PHV914-S the s 1 pg 0,054 1,599 97 1M 0.037 1.677 98 0.5 pg 0.066 1.672 96 0.5 pg SO 1,524 SO

 * χ

Φ χ * * * * «ψψ» Φ *

Φ Φ βΡβ * «β

To assess the stability of the NS3 / 4a epitope for performing the assay, the following assay is performed to determine the temporal variation in NS3 / 4a immune reactivation at room temperature. Small aliquots of stock NS3 / 4a were allowed to stand at room temperature and frozen at the intervals shown in Table 8. Each vial was coated at the same time and tested against two early NS3 seroconversion panels.

As shown in Table S, the NS3 / 4a stock solution is unstable and decreases in immunoreactivity over time. In addition, the NS3 / 4a conformation is required for immunoreactivity.

Further stability studies were performed as follows. Two conformational monoclonal antibodies directed against NS3 / 4a were replaced in anti-HCV early seroconversion panels. Ampoules of NS3 / 4a stock solution were stored at room temperature for 3, 6 and 2 hours. NS3 / 4a from frozen ampoules was used for coating in machine training at 90 ng / ml and analyzed by the procedure described above. The results suggest that the two monoclonal antibodies are indeed conformational and their reactivity is sensitive to treatment of NS3 / 4a at room temperature. The reactivity of a positive control monoclonal antibody did not change.

- · yflSj? S ** * ΦΦ. *? * 8 Λ- J φ «Χφ *

s / co S / C0 s / co / co s / co s / co s / eo s / co FHV 904-1 0.0 0.0 0.0 Ο, Ο 0.0 0.0 Ο, Ο 0.0 FHV 904-2 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 PHV 904-3 1.5 0.3 0.1 0.0 0.0 0.0 0.0 1.8 FHV 904-4 . 3.7 L0 0.2 o, i 0.1 0.1 0.1 4,4,4: FHV 904-5 4.8 0.7 0.0 0.3 0.2 0.3 44 5.5 FHV 904-6 • 5,4 2.3 Μ 4 4> Ö 0.6 0.5 0.6 5> S FHV 904-7 541 3.4 1.5 1.0 Μχ, 'Γ4 0.5 0.7 5.4 PHV 914-1 He / she 0.0 0.0 0.0 0.0 Ο, Ο 0.0 0.0 ^: FHV 914-2 0.0 0.0 Ο, Ο 0.0 0.0 0.0 0.0 0.0 PHV 914-3 Ο, Ο 0.0 0.0 0.0 0.0 0.0 0.0 Ö 0 PHV 914-4 0.5 0.1 0.0 0.0 0.0 0.0 0.0 0.7 FHV 914-5 2.1 0.4 0.0 0.0 0.0 0.0 0.0 3,0.4 PHV 914-6 2.3: 0.4 0.0 0.0 0.0 0.0 0.0 , · ³ > ⁴

Example 6

The conformational epitope of N83 / 4a against Imununrea NS3 / 4a

The immunoreactivity of the NS3 / 4a mimic epitope prepared as described above is compared with the immunoreactivity of N83 / 4a which is denatured by the addition of SDS to the NS3 / 4a conformational epitope composition at a final concentration of 2%. Denatured NS3 / 4a and conformational NS3 / 4a are used to coat microtiter plates as described above. The c206 antigen (Hepatology 15, 19-25 (1992) available from the ORTHO HCV version 3.0 ELISA Test System (Ortho-Clinical Diagnostics, Raritan, New Jersey)) was used to coat microtiter plates. The c20O antigen is used for comparison because it is assumed to be non-conforming because of the presence of reducing agent (OTT) and detergent (SDS) in its composition.

Immunoreactivity was tested against two early seroconversion panels, PHV 904 and PHV 914 (commercially available from human blood samples, manufactured by Bostor Biomedica, Inc., West Bridgewater, MA). The results are shown in Figure 9.

as shown in the denatured table. The data suggest that the denatured or linearized form of NS3 / 4a (as well as c250) does not detect early seroconversion panels as early as the conformational epitope of NS3 / 4a.

53 / 4a

2% SDS price

NS3 / 4a vs. denatured * tusk 2 ^v / o5US az j _s | For NS3 / 4a stock solution

NS3 Tc20F7 ~] NS3 | dNS3 j c200 j 'y 4a j / 4a * J í / 4 a

Γ ~ ο5 Γοπ / 4 a *> D j s / co j s / co I s / co | [0.012 1 0.012 ΤΟ, οδοΊ | 0 / D2H oM ™ fo ', 01 ~ |

0 / 0TF “OfoOóT | 0.02 ί'ο ^ ΐΐαοΓΐ j 1,124 I 0.071 | 0,045

b "" i 1 Ί ö / 07 "'j

2,401 | 0.273 | 0.129 j | 3.85 j 0.44 0.21

3.022 (0/793 j 0.347 | j 4.35 | 1.2S) 0.57 j

Sv '| 2,711 + 1,472 | 0.774 | | 4.85 1 2.87 Γΐ / 2βΊ j PHV] 3 _f 294 j 1,360 '' Ρ, ξ

1904-7 | | |

FHV 914-1 0,006 0,004 0,001 0.01 0.01 uh, 00 FHV 914-2 0,005 0,004 0,002 0.01 0.01 0.00 PHV 914-3 0.098 0,003 0,001 0.16 0.00 0.00 FHV 914-4 1,118 0,006 0,004 1.79 0.01 0.01 FHV 914-5 2,035 0,044 0,022 0.07 0.04 FHV 914-6 2.092 0.074 0,026 3.35 0.12 0.04 FHV 914-7 2.519 0.281 0432 S.Ö4 0:45 0.22 FHV 914-8 2.746 0.907 0,500 , 1:46 0.82 FHV 914-9 3,0S4 1,730 0.931 4.94 ' 2 ',? S 1.53 HCV 3.0 Neg. Kent. 0.023 0,024 0,008 conc blinds Neg. conc 0,027 0,024 0,007 Neg. Kosi 0,021 0,017 0,005 average 0,024 0,022 0.007:

Λ Μ *

cutting value 0,624 .622 .607 pose. contact 1,230 0.903 0.575 1.99 1.45 0.95 pose. lanyard. 1,445 0,9X6 .614 2.32 1.47 1.01

Immunoreactivity of the conformational epitope was also assayed using standard monoclonal antibodies against N83 / 4a. These monoclonal antibodies are then tested with BLXSA against NS3 / 4a and denatured NS3 / 4a and c250 antigen. The data show that anti-NS3 / 4a monoclonal antibodies react similarly to NS3 / 4a. and denatured NS3 / 4a as the eye conversion panels shown in Table 10. This is the result of that. also provides further evidence that the nature of MS3 / 4a is conformational since monoclonal antibodies with reactivity similar to those of early c33c saeroconversion panels can be prepared.

Γ Disc

N83 / 4 & dNS3 / 4a c200

L______ j MmMonal OD OD ob | 4B9 / B3 xoiöö 1,820 0.616 0.369 1 X: 1000 1,397 0.380 0.246

ί *

So we developed a new HCV detection assay. It will be apparent from the foregoing that, although specific embodiments of the present invention have been described by way of illustration, various modifications may be made therein without departing from the spirit and scope of the description herein.

Claims (32)

PATENT CLAIMS An immunoassay solid support comprising at least one hepatitis C virus (HCV) anti-core antibody and at least one isolated HCV NS3 / 4a epitope, wherein said NS3 / 4a epitope comprises the amino &lt; RTI ID = 0.0 &gt; 2, The solid support of claim 1 comprising at least two HCV anti-core antibodies bound. The solid support of claim 1, wherein at least one of the anti-core antibodies is directed to the Η-terminal region of the HCV core antigen. The solid carrier of claim 3, wherein at least one anti-core antibody is directed against amino acids 10-53 of HCV, a. By numbering corresponding to the HCV1 polyprotein sequence. The immunoassay solid carrier of claim 1, wherein said at least one anti-core antibody is a monoclonal antibody. The immunoassay solid support of claim 1, further comprising a multiple epitope fusion antigen bound. f. THE The immunoassay solid support of claim 7, wherein said multiple epitope fusion antigen comprises 7A-7F. 3A to 4A. Φ * Λ Φ 8, Immune solid support comprising two hepatitis C virus (HCV) anti-core monoclonal antibodies, a conformational epitope HCV NS3 / 4a linked to the amino acid sequence depicted in Figure 4A-4D, and a multiple epitope a fusion antigen comprising the sequence of 7A-7F. 4A to 6B. 9. A method for detecting hepatitis C virus infection in a biological sample, comprising the steps of: a) providing an immunoassay solid support according to claim 1; b) combining a biological sample with the solid support under conditions that allow the HCV antigens and antibodies, if present in the biological sample, to bind to at least one anti-core antibody or epitope NS3 / 4a; c) adding, under complexing conditions, to the solid support from step b) i) a first detectably labeled antibody, wherein the first labeled anti-core antibody is a detectably labeled HCV anti-core antibody, wherein said labeled anti-core antibody is an HCV core epitope which is different from at least one of the anti-core antibodies bound to the solid support; ii) an antigen that reacts with an HCV antibody from a biological sample reactive with an NS3 / 4a epitope; and iii) a second detectably labeled antibody, the second detectably labeled antibody being capable of reacting with the antigen of (ii); and d) detecting complexes between antibodies and antigens, if any, indicating that the biological sample is infected with HCV. 10. The method of claim 9, wherein said at least one anti-core antibody is directed against the N-terminal region of the HCV core antigen and said detectably labeled HCV antigen is a C-terminal region of the HCV core antigen. against. The method of claim 10, wherein at least one anti-core antibody is directed against amino acids 10-53 of HCV according to the numbering corresponding to the HCV1 polyprotein sequence, and said detectably labeled HCV anti-core antibody is HCV. It is directed against the amino acid residue 30 in the HCV1 polyprotein sequence. The method of claim 9, wherein said antigen, which reacts with an HCV antibody in the biological sample, comprises an epitope of the HCV polyprotein from the c33e region. 13. The method of claim 12, wherein the c33c epitope is fused to a human superoxide dismutase (hSÖD) amino acid sequence and the second detectably labeled antibody reacts with said hSOD amino acid sequence. 14, A method for detecting hepatitis C virus infection in a biological sample, comprising the steps of: a) providing an immunoassay solid support according to claim 2: b) combining a biological sample with the solid support under conditions that allow the HCV antigens and antibodies, if present in the biological sample, to bind to at least two of said anti-nuclear antibodies or the NS3 / 4a epitope; (c) complex training. adding to the solid support from step b) i) a first detectably labeled antibody, wherein the first detectably labeled antibody is a detectably labeled HCV anti-core antibody, and said labeled anti-core antibody is directed against an HCV core epitope, other than at least two anti-core antibodies bound to the solid support; ii) an epitope from the c33c region of the HCV polyprotein fused to an hSOD amino acid sequence; and iii) a second detectably labeled antibody, said second detectably labeled antibody being capable of reacting with said hSOD amino acid sequence; and d) detecting complexes between antibodies and antigens, if any, indicating that the biological sample is infected with HCV. The method of claim 14, wherein said at least two anti-core antibodies are directed against the N-terminal region of the HCV core antigen and said detectably labeled HCV anti-core antibody is a C-core HCV antigen. terminal region, 16. The method of claim 15, wherein at least two anti-core antibodies are directed against HCV amino acids 10-53, numbered according to the HCV1 polyprotein sequence, and said detectably labeled HCV anti-core antibody is a. HCV 12088 It is directed against amino acids 130 by numbering corresponding to the HCV1 polyprotein sequence. 17. A method for detecting hepatitis virus infection in a biological sample comprising the steps of: a) providing an immunoassay solid support according to claim 6; b) combining a biological sample with the solid support under conditions that allow the HCV antigens and antibodies, if present in the biological sample, to bind to at least one of said anti-nuclear antibodies, said NS3 / 4a epitope and said multiple fusion antigen; c) under complexing conditions, adding to the solid support from step b) i) a first detectably labeled antibody, wherein the first detectably labeled antibody is a detectably labeled HCV anti-core antibody and said labeled anti-core antibody against an HCV core epitope which is different from at least one anti-core antibody bound to the solid support; ii) first and second antigens which react with the HCV antibody in the biological sample, which reacts with said NS3 / 4a epitope and said multiple epitope fusion antigen; and ii) a second detectably labeled antibody, the second detectably labeled antibody being capable of reacting with antigens a ii); d) detecting complexes between antibodies and antigens, if any, indicating that the biological sample is infected with HCV. 18. The method of claim 17, wherein said at least one anti-core antibody is an N-terminus of the HCV core antigen * * * * φ φ · Χ # Φ Φ φ Φ Φ Φ * X ΦΦ> 'X is directed against and said first, detectably labeled. The HCV anti-core antibody is directed against the C-terminal region of HCV core antigen 19. The method of claim 18, wherein the at least one anti-core antibody is a. It is directed against HCV 1-5-5 amino acids by numbering corresponding to the H.CV1 polyprotein sequence, and said detectably labeled HCV anti-core antibody is directed against HCV 120130 amino acids by HCV1 polyprotein sequence numbering. 20. The method of claim 17, wherein said first antigen, which reacts with an HCV antibody in a biological sample, comprises an epitope derived from the c33c region of the HCV polyprotein. 21. The method of claim 20, wherein the c33c epitope is fused to a human superoxide dismutase (hSOD) amino acid sequence and the second detectably labeled antibody reacts with said hSOD amino acid sequence. 22. The method of claim 1.7, wherein said second antigen, which reacts with an HCV antibody in the biological sample, comprises an epitope on the c22 regions of the HCV polyprotein. 23. The method of claim 22, wherein the epitope derived from the c22 region comprises the Lysio-Sem amino acids of the HCV polyprotein, with the excision of Arg47 and. Leu. By replacing it with Trp at position 44, numbered based on the HCV1 polyprotein sequence, said epitope is targeted to a human super-disdismutose (hSOD) amino acid sequence and the second detectably labeled antibody reacts with said hSOD amino acid sequence. 24. The method of claim 17, wherein said multiple fusion antigen comprises the sequence of 7A-7F. The amino acid sequence shown in FIG. 25. A method of detecting hepatitis C virus infection in a biological sample, the method comprising the following plagues; a) providing an immunoassay solid support according to claim 3; b) combining a biological sample with the solid support under conditions that allow the HCV antigens and antibodies, if present in the biological sample, to bind to at least two of said anti-nuclear antibodies, said NS3 / 4a. a conformational epitope and said multiple fusion antigen; c) under complexing conditions, adding to the solid support from step h) i) a first detectably labeled antibody, wherein the first detectably labeled antibody is a detectably labeled HCV antibody, and said labeled anti-antibody against an HCV core epitope which is different from at least two anti-core antibodies bound to the solid support; ii) an epitope from the c33c region of the HCV polyprotein fused to the hSOD amino acid sequence and an epitope from the c22 region of the HCV polyprotein fused to the hSOD amino acid sequence; and sat) a second detectably labeled antibody which is a replicate -Φ, ΦΦΦΦ χ.Φ φ '# ΦΦ ♦' φ φ φ φ φ φ-, φ φ φ φφΦ Φί-Sft ΦΦ * $ detectably labeled antibody can react with said hSÖD aami, nosic acid sequence. sequences, respectively; detecting complexes between antibodies and antigens, if any, indicating that the biological sample is infected with HCV. 26, The method of claim 25, wherein said at least two anti-core antibodies are directed against the N-terminal region of the HCV core antigen, and said detectably labeled HCV anti-core antibody is the HCV core antigen. antigen C-terminal region 27, The method of claim 26, wherein said at least two anti-core antibodies to amino acids 10-53 of HCV are directed by the numbering corresponding to the HCV1 polyprotein sequence, and said detectably labeled HCV anti-core antibody is HCV. It is directed against amino acids 12013O, numbered according to the HCV1 polyprotein sequence. 28. The method of claim 25, wherein the epitope derived from the o22 region comprises the lysw-Serw amino acids of the HCV polyprotein, excised by Arg47 and replaced with Leu Trp at position 44, numbered based on the HCV1 polyprotein sequence. . 29. Immune Prosthetic Test Kit, including kit 1-8. An immunoassay solid support according to any one of claims 1 to 9 and instructions for performing an immunoassay test 92 A process for preparing an immunoassay solid support comprising the steps of; (a) providing a solid support; and b) binding at least one hepatitis C virus (HCV) anti-core antigen and an isolated conformation epitope HCV N83 / 4a comprising said amino acid sequence shown in Figures 4ekvenc-4Α. 31. A process for the preparation of an immunoassay solid support comprising the steps of: (a) providing a solid support; and b) binding of two hepatitis C virus (HCV) anti-core antigens and an isolated HCV NS3 / 4a conformational epitope comprising said amino acid sequence shown in Figures 4A-4D. The method of any one of claims 30 or 31, wherein at least one is applied to a solid support.